Publications by authors named "Kirk J Mantione"

34 Publications

Morphine stimulates nitric oxide release in human mitochondria.

J Bioenerg Biomembr 2015 Oct 9;47(5):409-17. Epub 2015 Sep 9.

MitoGenetics Research Institute, MitoGenetics LLC, 3 Bioscience Park Drive, Suite 307, Farmingdale, NY, 11735, USA.

The expression of morphine by plants, invertebrate, and vertebrate cells and organ systems, strongly indicates a high level of evolutionary conservation of morphine and related morphinan alkaloids as required for life. The prototype catecholamine, dopamine, serves as an essential chemical intermediate in morphine biosynthesis, both in plants and animals. We surmise that, before the emergence of specialized plant and animal cells/organ systems, primordial multi-potential cell types required selective mechanisms to limit their responsiveness to environmental cues. Accordingly, cellular systems that emerged with the potential for recruitment of the free radical gas nitric oxide (NO) as a multi-faceted autocrine/paracrine signaling molecule, were provided with extremely positive evolutionary advantages. Endogenous morphinergic signaling, in concert with NO-coupled signaling systems, has evolved as an autocrine/paracrine regulator of metabolic homeostasis, energy metabolism, mitochondrial respiration and energy production. Basic physiological processes involving morphinergic/NO-coupled regulation of mitochondrial function, with special emphasis on the cardiovascular system, are critical to all organismic survival. Key to this concept may be the phenomenon of mitochondrial enslavement in eukaryotic evolution via endogenous morphine.
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http://dx.doi.org/10.1007/s10863-015-9626-8DOI Listing
October 2015

Comparing bioinformatic gene expression profiling methods: microarray and RNA-Seq.

Med Sci Monit Basic Res 2014 Aug 23;20:138-42. Epub 2014 Aug 23.

Neuroscience Research Institute, State University of New York, College at Old Westbury, Old Westbury, USA.

Understanding the control of gene expression is critical for our understanding of the relationship between genotype and phenotype. The need for reliable assessment of transcript abundance in biological samples has driven scientists to develop novel technologies such as DNA microarray and RNA-Seq to meet this demand. This review focuses on comparing the two most useful methods for whole transcriptome gene expression profiling. Microarrays are reliable and more cost effective than RNA-Seq for gene expression profiling in model organisms. RNA-Seq will eventually be used more routinely than microarray, but right now the techniques can be complementary to each other. Microarrays will not become obsolete but might be relegated to only a few uses. RNA-Seq clearly has a bright future in bioinformatic data collection.
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http://dx.doi.org/10.12659/MSMBR.892101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4152252PMC
August 2014

Vascular dysfunction associated with type 2 diabetes and Alzheimer's disease: a potential etiological linkage.

Med Sci Monit Basic Res 2014 Aug 1;20:118-29. Epub 2014 Aug 1.

Neuroscience Research Institute, State University of New York - College at Old Westbury, Old Westbury, USA.

The endothelium performs a crucial role in maintaining vascular integrity leading to whole organ metabolic homeostasis. Endothelial dysfunction represents a key etiological factor leading to moderate to severe vasculopathies observed in both Type 2 diabetic and Alzheimer's Disease (AD) patients. Accordingly, evidence-based epidemiological factors support a compelling hypothesis stating that metabolic rundown encountered in Type 2 diabetes engenders severe cerebral vascular insufficiencies that are causally linked to long term neural degenerative processes in AD. Of mechanistic importance, Type 2 diabetes engenders an immunologically mediated chronic pro-inflammatory state involving interactive deleterious effects of leukocyte-derived cytokines and endothelial-derived chemotactic agents leading to vascular and whole organ dysfunction. The long term negative consequences of vascular pro-inflammatory processes on the integrity of CNS basal forebrain neuronal populations mediating complex cognitive functions establish a striking temporal comorbidity of AD with Type 2 diabetes. Extensive biomedical evidence supports the pivotal multi-functional role of constitutive nitric oxide (NO) production and release as a critical vasodilatory, anti-inflammatory, and anti-oxidant, mechanism within the vascular endothelium. Within this context, we currently review the functional contributions of dysregulated endothelial NO expression to the etiology and persistence of Type 2 diabetes-related and co morbid AD-related vasculopathies. Additionally, we provide up-to-date perspectives on critical areas of AD research with special reference to common NO-related etiological factors linking Type 2 diabetes to the pathogenesis of AD.
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http://dx.doi.org/10.12659/MSMBR.891278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4138067PMC
August 2014

Convergent dysregulation of frontal cortical cognitive and reward systems in eating disorders.

Med Sci Monit 2013 May 10;19:353-8. Epub 2013 May 10.

Center for Molecular and Cognitive Neuroscience, 1st Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.

A substantive literature has drawn a compelling case for the functional involvement of mesolimbic/prefrontal cortical neural reward systems in normative control of eating and in the etiology and persistence of severe eating disorders that affect diverse human populations. Presently, we provide a short review that develops an equally compelling case for the importance of dysregulated frontal cortical cognitive neural networks acting in concert with regional reward systems in the regulation of complex eating behaviors and in the presentation of complex pathophysiological symptoms associated with major eating disorders. Our goal is to highlight working models of major eating disorders that incorporate complementary approaches to elucidate functionally interactive neural circuits defined by their regulatory neurochemical phenotypes. Importantly, we also review evidence-based linkages between widely studied psychiatric and neurodegenerative syndromes (e.g., autism spectrum disorders and Parkinson's disease) and co-morbid eating disorders to elucidate basic mechanisms involving dopaminergic transmission and its regulation by endogenously expressed morphine in these same cortical regions.
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http://dx.doi.org/10.12659/MSM.889133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659002PMC
May 2013

Parkinson's disease, L-DOPA, and endogenous morphine: a revisit.

Med Sci Monit 2012 Aug;18(8):RA133-137

Neuroscience Research Institute, State University of New York - College at Old Westbury, Old Westbury, NY, USA.

Clinical observations stemming from widespread employment of restorative L-3,4-dihydroxyphenylalanine (L-DOPA) therapy for management of dyskinesia in Parkinson's Disease (PD) patients implicate a regulatory role for endogenous morphine in central nervous system dopamine neurotransmission. Reciprocally, it appears that restorative L-DOPA administration has provided us with a compelling in vivo pharmacological model for targeting peripheral sites involved in endogenous morphine expression in human subjects. The biological activities underlying endogenous morphine expression and its interaction with its major precursor dopamine strongly suggest that endogenous morphine systems are reciprocally dysregulated in PD. These critical issues are examined from historical and current perspectives within our short review.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3560700PMC
http://dx.doi.org/10.12659/msm.883259DOI Listing
August 2012

Neuroimmune-vascular cells and their pathological disorders.

Authors:
Kirk J Mantione

Arch Med Sci 2011 Oct 8;7(5):741-3. Epub 2011 Nov 8.

Neuroscience Research Institute, State University of New York Old Westbury, Old Westbury, NY, USA.

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http://dx.doi.org/10.5114/aoms.2011.25545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258799PMC
October 2011

Identification of a µ opiate receptor signaling mechanism in human placenta.

Med Sci Monit 2010 Nov;16(11):BR347-52

Neuroscience Research Institute, State University of New York, College at Old Westbury, Old Westbury, NY, USA.

Background: Previous studies report that genes in the morphine biosynthetic pathway have been found in placental tissue. Prior researchers have shown that kappa opioid receptors are present in human placenta. We determined if a µ opiate receptor was present and which subtype was expressed in human placenta. We also sought to demonstrate a functional µ opiate receptor in human placenta.

Material/methods: Polymerase chain reactions as well as DNA sequencing were performed to identify the µ opiate receptor subtypes present in human placenta. The functionality of the receptor was demonstrated by real time amperometric measurements of morphine induced NO release.

Results: The µ4 opiate receptor sequence was present as well as the µ1 opioid receptor transcript. The addition of morphine to placental tissue resulted in immediate nitric oxide release and this effect was blocked by naloxone.

Conclusions: In the present study, an intact morphine signaling system has been demonstrated in human placenta. Morphine signaling in human placenta probably functions to regulate the immune, vascular, and endocrine functions of this organ via NO.
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November 2010

Catechol-O-methyltransferase: potential relationship to idiopathic hypertension.

Arch Med Sci 2010 Jun;6(3):291-5

Neuroscience Research Institute, State University of New York - College at Old Westbury, New York, USA.

Catecholamine signaling pathways in the peripheral and central nervous systems (PNS, CNS, respectively) utilize catechol-O-methyltransferase (COMT) as a major regulatory enzyme responsible for deactivation of dopamine (DA), norepinephrine (NE) and epinephrine (E). Accordingly, homeostasis of COMT gene expression is hypothesized to be functionally linked to regulation of autonomic control of normotensive vascular events. Recently, we demonstrated that morphine administration in vitro resulted in decreased cellular concentrations of COMT-encoding mRNA levels, as compared to control values. In contrast, cells treated with E up regulated their COMT gene expression. In sum, these observations indicate a potential reciprocal linkage between end product inhibition of COMT gene expression by E and morphine. Interestingly, the observed effects of administered E on COMT gene expression suggest an enhancement of its own catabolism or, reciprocally, a stimulation morphine biosynthesis.
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http://dx.doi.org/10.5114/aoms.2010.14246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282503PMC
June 2010

Identification of endogenous morphine and a mu3-like opiate alkaloid receptor in human brain tissue taken from a patient with intractable complex partial epilepsy.

Med Sci Monit 2008 Jun;14(6):CS45-49

Department of Psychiatry, Division of Psychiatry and Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.

Background: We set out to detect whether morphine is present in tissue taken from a patient with intractable temporal lobe epilepsy and to characterize the presence and nature of mu opiate receptor subtypes in this tissue.

Case Report: In temporal lobe tissue, resected during anteromedial temporal lobectomy for intractable focal epilepsy, morphine was identified by quantitative radioimmunoassay (RIA) coupled to electrochemical detection via high-pressure liquid chromatography (HPLC). In addition, RNA isolated from the medial and lateral temporal lobe specimens was analyzed by conventional and real time reverse transcriptase-polymerase chain reaction (RT-PCR) for the expression of different human receptor gene transcripts. RIA revealed the presence of morphine at 3.4 nanograms per gram of tissue wet weight. Using RT-PCR and a primer specifically set for the mu3 (550 base pair fragment) and mu4 (880 base pair fragment) MOR splice variants, a mu4 splice variant was identified in both brain sections.

Conclusions: This human brain tissue study of a subject with temporal lobe epilepsy documents the presence of endogenous morphine and of a mu4 splice variant. These findings may have implications for our understanding of the mechanism of temporal lobe epilepsy.
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June 2008

Estrogen's actions transcend a sole reproductory function in cell signaling.

Authors:
Kirk J Mantione

Med Sci Monit 2008 Feb;14(2):SC1-3

Neuroscience Research Institute, State University of New York, College at Old Westbury, Old Westbury, NY 11568-0210, USA.

A literature has been emerging showing that estrogen exhibits functions that transcends its role in reproduction. 17beta-estradiol stimulates nitric oxide (NO) release from human internal thoracic artery fragments and from cultured arterial endothelial cells by acting on an estrogen cell surface receptor. Estrogen down regulates immunocyte functions, i.e. chemotaxis and phagocytosis, as well. Monocytes express estrogen receptor mRNA as well as an estrogen receptor binding site. Importantly, estrogen exerts non reproductive roles in invertebrates. These reports also demonstrate that invertebrate estrogen receptors are coupled to constitutive NO release and are located on the cell surface, suggesting they first appear very early in evolution. All in all, estrogen's ability to stimulate constitutive nitric oxide synthase derived NO is significant since NO is also considered an important inhibitory agent that diminishes immunocyte adhesion and the vascular endothelium's capability to adhere immunocytes, as well as down regulating various immunocytes both before and after proinflammatory events. These findings promise to open up new areas of investigation concerning estrogen associated biomedical phenomena, including cellular protection processes.
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February 2008

Endogenous morphine/nitric oxide-coupled regulation of cellular physiology and gene expression: implications for cancer biology.

Semin Cancer Biol 2008 Jun 8;18(3):199-210. Epub 2007 Dec 8.

Neuroscience Research Institute, State University of New York - SUNY College at Old Westbury, P.O. Box 210, Old Westbury, NY 11568, USA.

Cancer is a simplistic, yet complicated, process that promotes uncontrolled growth. In this regard, this unconstrained proliferation may represent primitive phenomena whereby cellular regulation is suspended or compromised. Given the new empirical evidence for a morphinergic presence and its profound modulatory actions on several cellular processes it is not an overstatement to hypothesize that morphine may represent a key chemical messenger in the process of modulating proliferation of diverse cells. This has been recently demonstrated by the finding of a novel opiate-alkaloid selective receptor subtype in human multilineage progenitor cells (MLPC). Adding to the significance of morphinergic signaling are the findings of its presence in plant, invertebrate and vertebrate cells, which also have been shown to synthesize this messenger as well. Interestingly, we and others have shown that some cancerous tissues contain morphine. Furthermore, in medullary histolytic reticulosis, which is exemplified by cells having hyperactivity, the mu3 (mu3) opiate select receptor was not present. Thus, it would appear that morphinergic signaling has inserted itself in many processes taking a long time to evolve, including those regulating the proliferation of cells across diverse phyla.
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http://dx.doi.org/10.1016/j.semcancer.2007.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2432462PMC
June 2008

Persistence of evolutionary memory: primordial six-transmembrane helical domain mu opiate receptors selectively linked to endogenous morphine signaling.

Med Sci Monit 2007 Dec;13(12):SC5-6

Neuroscience Research Institute, State University of New York, College at Old Westbury, Old Westbury, NY 11568-0210, USA.

Biochemical, molecular and pharmacological evidence for two unique six-transmembrane helical (TMH) domain opiate receptors expressed from the micro opioid receptor (MOR) gene have been shown. Designated micro3 and micro4 receptors, both protein species are Class A rhodopsin-like members of the superfamily of G-protein coupled receptors but are selectively tailored to mediate the cellular regulatory effects of endogenous morphine and related morphinan alkaloids via stimulation of nitric oxide (NO) production and release. Both micro3 and micro4 receptors lack an amino acid sequence of approximately 90 amino acids that constitute the extracellular N-terminal and TMH1 domains and part of the first intracellular loop of the micro1 receptor, but retain the empirically defined ligand binding pocket distributed across conserved TMH2, TMH3, and TMH7 domains of the micro1 sequence. Additionally, the receptor proteins are terminated by unique intracellular C-terminal amino acid sequences that serve as putative coupling or docking domains required for constitutive NO synthase activation. Because the recognition profile of micro3 and micro4 receptors is restricted to rigid benzylisoquinoline alkaloids typified by morphine and its extended family of chemical congeners, it is hypothesized that conformational stabilization provided by interaction of extended extracellular N-terminal protein domains and the extracellular loops is required for binding of endogenous opioid peptides as well as synthetic flexible opiate alkaloids.
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December 2007

A functionally coupled mu3-like opiate receptor/nitric oxide regulatory pathway in human multi-lineage progenitor cells.

J Immunol 2007 Nov;179(9):5839-44

Neuroscience Research Institute, State University of New York College, Old Westbury, NY 11568, USA.

Ongoing studies from our group support the existence and biological importance of a distinct cellular signaling pathway involving endogenously synthesized, chemically authentic, l-morphine, its cognate mu(3) opiate receptor subtype, and constitutive NO synthase. Based on prior studies indicating evolutionary conservation and adaptation of morphinergic/NO-coupled signaling to mediate autocrine/paracrine control of cellular functions, our goal was to determine whether a functionally competent mu(3) opiate receptor/NO-coupled regulatory pathway exists in human multilineage progenitor cells (MLPC) prepared from umbilical cord blood. Real-time PCR analysis indicated significant expression of mu(3) opiate receptor-encoding RNA by undifferentiated human MLPC, in the absence of traditional mu(1) opioid receptor-encoding RNA expression. Unpredictably, confirmatory RT-PCR analyses indicated cellular expression of a splice variant of the previously characterized mu(3) opiate receptor-encoding mRNA. Pharmacological analyses provided critical validating evidence of functional mu(3)-like opiate receptor/NO-coupled signaling within primary cultures of undifferentiated human MLPC via morphine-evoke real-time release of NO. Control analyses indicated that morphine-stimulated NO release was markedly inhibited by prior treatment with the opiate antagonist l-naloxone or the constitutive NO synthase inhibitor N(G)-nitro-l-arginine methyl ester and unresponsive to stimulation by the opioid peptide methionine enkephalin. Complementary microarray analysis demonstrated that traditional mu(1), delta, and kappa opioid receptor gene expression is not detected in both undifferentiated and differentiated MLPC. Chemical differentiation of MLPC into neuronal progenitor cells effected significant phenotypic expression of a variety of neurally-associated genes. Our data provide compelling evidence in support of both the evolutionary primacy and primordial regulatory role of mu(3)-like opiate receptor/NO signaling in embryogenesis.
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http://dx.doi.org/10.4049/jimmunol.179.9.5839DOI Listing
November 2007

Endogenous morphine signaling via nitric oxide regulates the expression of CYP2D6 and COMT: autocrine/paracrine feedback inhibition.

Addict Biol 2008 Mar 16;13(1):118-23. Epub 2007 Jun 16.

Neuroscience Research Institute, State University of New York-College at Old Westbury, 11568, USA.

We determined changes in mRNA expression in specific enzymes involved in the biosynthesis of morphine in human white blood cells via microarray. Leukocyte exposure to morphine down-regulated catechol-O-methyl transferase (COMT) and CYP2D6 by approximately 50% compared with control values. The treatment did not alter DOPA decarboxylase and dopamine beta-hydroxylase expression, demonstrating the specificity of morphine actions. The verification of the microarray data was accomplished via real-time Taqman reverse transcriptase polymerase chain reaction (RT-PCR) focused on CYP2D6 and COMT expression in different blood samples treated with morphine. The analysis showed similar changes in the expression of CYP2D6 and COMT mRNA. The expression was reduced by 47 +/- 7% for CYP2D6, substantiating the microarray finding of a 54% reduction. Furthermore, exposure of white blood cells to 10(-6) M S-nitroso-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, reduced the expression of CYP2D6 and COMT. Prior naloxone (10(-6) M) or N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) addition abrogated morphine's down-regulating activity, demonstrating morphine was initiating its actions via stimulating constitutive NO synthase derived NO release via the mu3 opiate receptor splice variant. In the past we demonstrated that UDP-glucurosyltransferase is involved in metabolizing morphine to morphine 6-glucuronide in adrenal chromaffin cells. In the present study its expression was not found in controls and morphine-treated cells, suggesting that morphine 6-glucuronide may not be synthesized in white blood cells. Taken together, it appears that morphine has the ability to modulate its own synthesis via autocrine and paracrine signaling.
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http://dx.doi.org/10.1111/j.1369-1600.2007.00072.xDOI Listing
March 2008

Nicotine, alcohol and cocaine coupling to reward processes via endogenous morphine signaling: the dopamine-morphine hypothesis.

Med Sci Monit 2007 Jun;13(6):RA91-102

Neuroscience Research Institute, State University of New York College at Old Westbury, Old Westbury, NY 11568, USA.

Pleasure is described as a state or feeling of happiness and satisfaction resulting from an experience that one enjoys. We examine the neurobiological factors underlying reward processes and pleasure phenomena. With regard to possible negative effects of pleasure, we focus on addiction and motivational toxicity. Pleasure can serve cognition, productivity and health, but simultaneously promotes addiction and other negative behaviors. It is a complex neurobiological phenomenon, relying on reward circuitry or limbic activity. These processes involve dopaminergic signaling. Moreover, nicotine, cocaine and alcohol appear to exert their pleasure providing action via endogenous morphinergic mechanisms. Natural rewarding activities are necessary for survival and appetitive motivation, usually governing beneficial biological behaviors like eating, sex and reproduction. Social contacts can further facilitate the positive effects exerted by pleasurable experiences. However, artificial stimulants can be detrimental, since flexibility and normal control of behavior are deteriorated. Additionally, addictive drugs are capable of directly acting on reward pathways, now, in part, via endogenous morphine processes.
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June 2007

Detection of nitric oxide in exhaled human breath: exercise and resting determinations.

Med Sci Monit 2007 Mar;13(3):MT1-5

Kiernan Wellness Center, Rockaway Park, NY 11694, USA.

Background: Nitric oxide has become a vital indicator of health since many cells produce it constitutively. It is present in exhaled breath and can be measured.

Material/methods: A Kiernan NO Breath analyzer (KNB) was used in the present study to determine nitric oxide (NO) levels in exhaled human breath. The KNB was calibrated via measuring NO gas in O2-free N2 obtained from Scott Specialty Gases. Human subjects aged 21 to 45 were instructed to place the KNB over their nose and mouth and to breathe normally before and after mild exercise (n=24) and relaxation (n=20). Mean exhaled NO measurements were compared before and after the protocols using paired t-tests.

Results: Regardless of the test, all subjects exhibited NO in their exhaled breath. Exhaled NO decreased significantly after exercise compared to the first reading just prior to the exercise protocol. The mean +/-SE of exhaled NO was 22.8+/-4 before and 13.0+/-2 ppb after exercise (n=24, P=0.003). In the resting experiment, exhaled NO was demonstrated to increase significantly after 10 min compared to the reading taken right after the individuals sat down.

Conclusions: The present study demonstrates NO in exhaled human breath can vary, reflecting the activity state of the individual. Additionally, the study demonstrates that NO in exhaled human breath can be measured rapidly, with high sensitivity, and in real time via the KNB, representing an affordable means to achieve this determination.
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March 2007

Nitric oxide's pulsatile release in lobster heart and its regulation by opiate signaling: pesticide interference.

Med Sci Monit 2006 Dec 23;12(12):BR373-378. Epub 2006 Nov 23.

Neuroscience Research Institute, State University of New York College at Old Westbury, Old Westbury, NY 11568, USA.

Background: Data is emerging in the human and invertebrate literature demonstrating that mu opiate receptors and morphine are present in cardiovascular tissues in diverse animals, including human tissues, where they may be exerting a cardioregulatory role via stimulation of constitutive nitric oxide (NO) production.

Material/methods: NO release from lobster heart was evaluated without stimulation and after morphine exposure using a real-time NO-specific amperometric probe. In addition, real time NO release was evaluated after treatment with low doses of widely used pesticides (e.g., pyrethroids). Real time RT-PCR was used to investigate the presence of mu opiate receptor subtypes in lobster heart.

Results: Basal NO release occurs in lobster heart at the nanomolar level. Morphine enhanced this level of release; naloxone (an opiate antagonist) blocked it, as did exposure to the NO synthase inhibitor L-NAME. In addition, treatment with the pyrethroids, permethrin and resmethrin, abrogated constitutive NO release from lobster heart. Finally, by way of real time RT-PCR we were able to demonstrate the presence of the micro(3) opiate receptor subtype in lobster heart.

Conclusions: Rhythmic NO bursts appear to be involved in normal cardiac muscle activity in Homarus americanus. Lobster heart contains morphinergic signaling components capable of slowing down its beating rate via NO production. In addition, compounds such as pyrethroid pesticides may alter normal cardiac activity by interfering with constitutive NO production and thus, depressing basal NO levels. This may ultimately make these animals more susceptible to environmental assaults/toxins.
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December 2006

Cholinergic regulation of endogenous morphine release from lobster nerve cord.

Med Sci Monit 2006 Sep;12(9):BR295-301

Neuroscience Research Institute, State University of New York-College at Old Westbury, P.O. Box 210, Old Westbury, NY 11568, USA.

Background: Invertebrate nervous systems are regulated by G-coupled protein receptors, chemical transporters, and ion channels responsive to established drugs of abuse including opiates, alcohol, psychostinulants, and nicotine. Thus, invertebrate nervous tissue preparations can be used as predictive model systems by which to evaluate underlying pharmacological mechanisms of addictive processes.

Material/methods: Ex vivo pharmacological trials were used to determine the comparative effects of the nicotinic agonists and antagonists on the evoked release of labeled morphine from H. americanus nerve cord. The intrinsically low basal levels of endogenous morphine required that we utilize an ex vivo model system involving pre-labeling of intracellutlar opiate alkaloid pools with high specific activity 125I labeled morphine.

Results: Both nicotine and epibatidine promoted evoked release of 125I labeled morphine that is selectively linked to activation of invertebrate nicotinic receptors based on pharmacological inhibition by alpha bungarotoxin (alpha-BuTx). Epibatidine promoted release at concentrations 2-3 orders of magnitude higher than nicotine. Co-administration of nicotine (60 nM) and the pre-junctional ganglionic nicotinic antagonist hexamethonium (1 microM) produced a marked potentiation of 125I labeled morphine release; a pharmacological effect also observed for epibatidine (35 microM) co-administered with the competitive nicotinic antagonist chlorisondaminie at 1 microM. The stimulatory effects of ethanol to promote enhanced release of endogenous morphine were not affected by co-admninistration of alpha-BuTx at 1 microM.

Conclusions: The stirmulatory effects of nicotine on cellular expression and release of endogenous morphine occurs via specific alpha-BuTx sensitive receptors, suggesting a novel mechanism underling the reinforcing and addictive properties of nicotine via endogenous morphine.
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September 2006

Morphine regulates gill ciliary activity via coupling to nitric oxide release in a bivalve mollusk: opiate receptor expression in gill tissues.

Med Sci Monit 2006 Jun 29;12(6):BR195-200. Epub 2006 May 29.

Neuroscience Research Institute, State University of New York-College at Old Westbury, Old Westbury, NY 11568, USA.

Background: Invertebrates express opiate receptors and synthesize opiate alkaloids such as morphine and morphine-6beta-glucuronide. Most of this work has been demonstrated in immune and neural tissues of various invertebrates. We hypothesized that morphinergic signaling may also take place in Mytilus edulis gill since they are innervated, in part, with dopamine nerves.

Material/methods: Ciliary activity from excised gills was evaluated via stroboscopic synchronization of metachronal wave formation before and after drug exposure. Nitric oxide was determined in real-time via an amperometric probe following drug application. Real-time RT-PCR was performed on excised gill tissue to confirm the presence of the mu opiate receptor transcript.

Results: Incubation of M. Edulis excised gill filaments reveal spontaneously lateral cilia beating in a metachronal wave of about 600 beats per minute, which was significantly decreased by morphine in a concentration dependent and naloxone reversible manner. Exposure of the spontaneously beating cilia to SNAP, a nitric oxide donor, also diminished the beating rate in a concentration dependent manner. Exposing the cilia to L-NAME blocked the morphine induced cilio-inhibition, demonstrating that morphine was working to inhibit the cilia via NO. Furthermore, the gill tissue contained mu opiate receptor transcripts, which was mu3 in nature.

Conclusions: As in mammals, opiate signaling is not confined to neural tissues. This report demonstrates the occurrence of opiate signaling for the first time in an invertebrate's respiratory tissue.
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June 2006

Norlaudanosoline and nicotine increase endogenous ganglionic morphine levels: nicotine addiction.

Cell Mol Neurobiol 2006 Jul-Aug;26(4-6):1037-45. Epub 2006 Apr 28.

Neuroscience Research Institute, State University of New York, College at Old Westbury, P.O. Box 210, Old Westbury, New York 11568, USA.

1. Given the presence of morphine, its metabolites and precursors, e.g., norlaudanosoline, in mammalian and invertebrate tissues, it became important to determine if exposing normal excised ganglia to norlaudanosoline would result in increasing endogenous morphine levels. 2. Mytilus edulis pedal ganglia contain 2.2 +/- 0.41 ng/g wet weight morphine as determined by high pressure liquid chromatography coupled to electrochemical detection and radioimmunoassay. 3. Incubation of M. edulis pedal ganglia with norlaudanosoline, a morphine precursor, resulted in a concentration- and time-dependent statistical increase in endogenous morphine levels (6.9 +/- 1.24 ng/g). 4. Injection of animals with nicotine also increased endogenous morphine levels in a manner that was antagonized by atropine, suggesting that nicotine addiction may be related to altering endogenous morphine levels in mammals. 5. We surmise that norlaudanosoline is being converted to morphine, demonstrating that invertebrate neural tissue can synthesize morphine.
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http://dx.doi.org/10.1007/s10571-006-9021-4DOI Listing
April 2007

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

Med Sci Monit 2006 May;12(5):BR155-61

Neuroscience Research Institute, State University of New York - College at Old Westbury, Old Westbury, NY 11568, USA.

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.
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May 2006

Human white blood cells synthesize morphine: CYP2D6 modulation.

J Immunol 2005 Dec;175(11):7357-62

Neuroscience Research Institute, State University of New York College at Old Westbury, Old Westbury, NY 11568, USA.

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.
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http://dx.doi.org/10.4049/jimmunol.175.11.7357DOI Listing
December 2005

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

Med Sci Monit 2005 Nov;11(11):BR397-404

Neuroscience Research Institute, State University of New York, College at Old Westbury, New York 11568, USA.

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.
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November 2005

Morphine modulation of the ubiquitin-proteasome complex is neuroprotective.

Med Sci Monit 2005 Nov;11(11):BR386-96

Neuroscience Research Institute, State University of New York Old Westbury, Old Westbury NY 11568, USA.

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.
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November 2005

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

Med Sci Monit 2005 Oct 26;11(10):BR357-66. Epub 2005 Sep 26.

Neuroscience Research Institute, State University of New York, College at Old Westbury, Old Westbury, NY 11568, USA.

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.
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October 2005

Morphine 6beta glucuronide: fortuitous morphine metabolite or preferred peripheral regulatory opiate?

Med Sci Monit 2005 May 28;11(5):MS43-46. Epub 2005 Apr 28.

Neuroscience Research Institute, State University of New York College at Old Westbury, Old Westbury, NY, USA.

Morphine-6beta-glucuronide (M6G), a metabolite of morphine that the brain can produce, is an opiate agonist that appears to have a greater analgesic potency than morphine. M6G has a 1-octanol/water partition coefficient 187 times lower than that of morphine and M6G has a blood brain barrier permeability 57 times lower than morphine. The brain uptake rate however is only 32 times lower, suggesting that an active transport mechanism might be present. Furthermore, evidence for a distinct receptor for M6G also appears to be emerging. Real time polymerase chain reactions allowed for the discovery of single nucleotide polymorphisms (SNP's) in the human mu opioid receptor gene. The most common SNP is a substitution at base118 where A is replaced with G (A118G). This SNP has a decreased potency for M6G in individuals possessing it whereas the potency of morphine is unaffected by this SNP. The possibility that a peripheral opiate signaling system, using M6G and its distinct receptor, exists seems plausible. Taken together, if a distinct M6G signaling mechanism does exist, the fact that morphine can be converted into a more water soluble compound that might be more potent would not be an accident.
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May 2005

In vivo and in vitro L-DOPA and reticuline exposure increases ganglionic morphine levels.

Med Sci Monit 2005 May 28;11(5):MS1-5. Epub 2005 Apr 28.

Neuroscience Research Institute, State University of New York College at Old Westbury, 11568, USA.

Background: Given the presence of morphine, its metabolites and precursors, i.e., reticuline, in mammalian and invertebrate tissues, it has become imperative to determine if exposing tissues to putative opiate alkaloid and dopamine precursors would result in increasing endogenous morphine levels.

Material/methods: Endogenous morphine levels were determined by high performance liquid chromatography coupled to electrochemical detection and radioimmunoassay, following incubation of Mytilus edulis pedal ganglia with reticuline or L-DOPA. Injection of L-DOPA or reticuline into healthy animals was via the foot.

Results: Ganglia incubated in vitro with reticuline or L-DOPA for 1 hour exhibited a concentration and time dependent statistically significant increase in their endogenous morphine levels (5.0 +/- 0.47, 3.6 +/- 0.45 ng/ganglion, respectively). Injection of intact, healthy animals with reticuline or L-DOPA also results in significantly higher endogenous ganglionic morphine levels.

Conclusions: Taken together, we show that L-DOPA is being converted to morphine, demonstrating that pedal ganglia can synthesize morphine from these putative precursors in vitro and in vivo. This is the first demonstration of morphine being synthesized in a normal, healthy free living animal.
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May 2005

Reticuline exposure to invertebrate ganglia increases endogenous morphine levels.

Neuro Endocrinol Lett 2004 Oct;25(5):323-30

Neuroscience Research Institute, State University of New York College at Old Westbury, NY 11568, USA.

Objectives: Given the presence of morphine, its metabolites and precursors in mammalian and invertebrate tissues, it became important to determine if exposing tissues to an opiate alkaloid precursor, reticuline, would result in increasing endogenous morphine levels.

Method: Endogenous morphine levels were determined by high pressure liquid chromatography coupled to electrochemical detection and radioimmunoassay following incubation of Mytilus edulis pedal ganglia with reticuline. Nitric oxide (NO) release was determined in real-time via an amperometric probe. Mu opiate receptor affinity for opiate alkaloid precursors was determined by a receptor displacement assay.

Results: Morphine is present in the pedal ganglia of Mytilus edulis (1.43 +/- 0.41 ng/mg +/- SEM ganglionic wet weight). Ganglia incubated with 50 ng of reticuline, a morphine precursor in plants, for 1 hour exhibited a statistical increase in their endogenous morphine levels (6.7 +/- 0.7 ng/mg tissue wet weight; P<0.01). This phenomenon is concentration dependent. The increase in ganglionic morphine levels occurs gradually over the 60 min incubation period, beginning 10 minutes post reticuline addition. We show that reticuline (10(-6) M) does not stimulate ganglionic NO release in a manner resembling that of morphine (10(-6) M), which releases NO seconds after its exposure to the ganglia and lasts for 5 minutes. With reticuline, there is a 3 minute delay, which is followed by an extended release period. Furthermore, in binding displacement experiments both reticuline and salutaridine (another morphine precursor) exhibit no binding affinity for the pedal ganglia mu opiate receptor subtype. This finding is further substantiated using the positive control of human monocytes where the mu3 opiate receptor subtype has been cloned.

Conclusion: Taken together, we surmise that the morphine's precursors are being converted to morphine. The experiments strongly indicate that pedal ganglia can synthesize morphine from reticuline.
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October 2004

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

Med Sci Monit 2004 Oct 23;10(10):BR351-5. Epub 2004 Sep 23.

Neuroscience Research Institute, State University of New York College at Old Westbury, Old Westbury, NY 11568, USA.

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.
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October 2004

Endogenous morphinergic signaling and tumor growth.

Front Biosci 2004 Sep 1;9:3176-86. Epub 2004 Sep 1.

Neuroscience Research Institute, State University of New York, College at Old Westbury, Old Westbury, NY 11568-0210, USA.

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.
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http://dx.doi.org/10.2741/1471DOI Listing
September 2004