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George B. Stefano - One of the best experts on this subject based on the ideXlab platform.
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Seasonal variations in Mu Opiate Receptor signaling in the nervous system of the blue Mussel, Mytilus edulis: temperature controls physiological processes
ISJ-Invertebrate Survival Journal, 2010Co-Authors: Kirk J. Mantione, Patrick Cadet, F Casares, Wei Zhu, George B. StefanoAbstract:It is anticipated that invertebrate processes will be subject to seasonal variations because of their poikilothermal characteristics. In the present study we determined if the morphine coupled nitric oxide (NO) release, which is constitutive in nature, exhibits seasonal characteristics, which has previously been shown for catecholamine processes in the marine mollusc Mytilus edulis. In this regard, morphine induced NO release measured on a monthly basis for one year revealed a peak release value (39 ± 4 nM) during the late spring and early summer. The lowest NO release occurred during the months of January (6.0 ± 0.5 nM) through March (6.5 ± 1.1 nM). The lowest sea surface temperatures (1.3 °C) were also recorded in these same three winter months in New York. Relative Mu Opiate Receptor gene expression was assessed by real time PCR during these seasons. The mRNA expression reached a relative peak during the month of June and was at its lowest in February and March, further demonstrating the direct coupling of morphine with this Receptor. We conclude that the temperature an animal is chronically exposed to serves to control cellular processes, i.e., Opiate signaling.
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Morphine regulates gill ciliary activity via coupling to nitric oxide releasein a bivalve mollusk: Opiate Receptor expression in gill tissues.
Medical science monitor : international medical journal of experimental and clinical research, 2006Co-Authors: Kirk J. Mantione, George B. Stefano, Celline KimAbstract: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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The American lobster, Homarus americanus, contains morphine that is coupled to nitric oxide release in its nervous and imMune tissues: Evidence for neurotransmitter and hormonal signaling.
Neuro endocrinology letters, 2005Co-Authors: Federico Casares, Kirk J. Mantione, Wei Zhu, Anne Mcelroy, Geert Baggermann, George B. StefanoAbstract:OBJECTIVES: The study was designed to determine if morphine was present in lobster tissues. It was also important to determine, as in other animals, if its levels would change in response to stress. In this regard, it was also important to determine if lobster imMune and neural tissues express the Mu Opiate Receptor subtype, which was coupled to constitutive nitric oxide synthase derived nitric oxide release. METHODS: Homarus americanus were used in these experiments. Morphine was purified in lobster tissues via high pressure liquid chromatography coupled to UV detection. It was quantified via radioimMunoassay (RIA) and was identified via quadruple time of flight - mass spectrometry. Animals were subject to 2 forms of trauma, namely pereiopod-ablation or lipopolysaccaride (LPS) - injection, and morphine levels determined in nerve cord or hemolymph. Real-time nitric oxide production was determined via an amperometric probe. RT-PCR was used to determine the presence of a micro Opiate Receptor transcript. RESULTS: In Homarus americanus hemolymph and nerve cord morphine was found. RIA revealed morphine levels of 3.36 pg/mg +/ - 0.48 SEM (N=8) in nerve cord and 717.88 pg/ml +/ - 56.77 SEM (N=58) in hemolymph. In stressed (pereiopod-ablated or LPS-injected) animals, the endogenous morphine levels initially increased significantly by 24% for hemolymph and 48% for nerve cord. By day 5, the stressed and control values for endogenous morphine, in both tissues, was lower and non-distinguishable. In both hemocytes and neural cells, morphine, not met-enkephalin, stiMulated constitutive nitric oxide release in a naloxone antagonizable manner, demonstrating a Mu Opiate Receptor mediated phenomenon and suggesting the presence of the Mu Opiate Receptor subtype, micro3, since it is Opiate alkaloid selective and opioid peptide insensitive. RT-PCR revealed the presence of a micro Opiate Receptor transcript in Homarus neural and imMune tissues, which exhibits a 100% sequence identity with its human counterpart. CONCLUSION: Taken together, after eliminating all sources of contamination, morphine is present in lobster tissues, potentially demonstrating hormonal and neurotransmitter functions that are involved in the animals' stress response.
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Differential expression of the human Mu Opiate Receptor from different primary vascular endothelial cells
Medical science monitor : international medical journal of experimental and clinical research, 2004Co-Authors: Patrick Cadet, Kirk J. Mantione, Thomas V. Bilfinger, George B. StefanoAbstract: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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Endogenous morphine: A role in wellness medicine
Medical science monitor : international medical journal of experimental and clinical research, 2004Co-Authors: George B. StefanoAbstract:The demonstration of the Multiplicity of Opiate Receptor types has led to the understanding that, depending on their site of action, opioid peptides as well as Opiate alkaloids may bind to more than one Opiate Receptor subtype. In addition to the two main Mu Opiate Receptor subtypes, Mu1 and Mu2, our laboratory has demonstrated a third Mu Opiate Receptor (Mu3) that is selective for Opiate alkaloids but insensitive to opioid peptides. Recently, the Mu3 Opiate Receptor subtype has been cloned from human imMune, vascular and neural tissues. This Mu3 story complements many biochemical reports; demonstrating morphine is an endogenous signaling molecule, functioning in the capacity of a neurotransmitter and hormone. Adding additional evidence to this hypothesis are the findings of morphine precursors in mammalian and invertebrate tissues. The reports published in this issue of MSM complement this story while advancing the hypothesis by placing Opiate alkaloid signaling in limbic structures. The pharmacological characteristics of exogenous morphine find a role for explaining morphine action in an "emotional" and belief setting.
Patrick Cadet - One of the best experts on this subject based on the ideXlab platform.
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Seasonal variations in Mu Opiate Receptor signaling in the nervous system of the blue Mussel, Mytilus edulis: temperature controls physiological processes
ISJ-Invertebrate Survival Journal, 2010Co-Authors: Kirk J. Mantione, Patrick Cadet, F Casares, Wei Zhu, George B. StefanoAbstract:It is anticipated that invertebrate processes will be subject to seasonal variations because of their poikilothermal characteristics. In the present study we determined if the morphine coupled nitric oxide (NO) release, which is constitutive in nature, exhibits seasonal characteristics, which has previously been shown for catecholamine processes in the marine mollusc Mytilus edulis. In this regard, morphine induced NO release measured on a monthly basis for one year revealed a peak release value (39 ± 4 nM) during the late spring and early summer. The lowest NO release occurred during the months of January (6.0 ± 0.5 nM) through March (6.5 ± 1.1 nM). The lowest sea surface temperatures (1.3 °C) were also recorded in these same three winter months in New York. Relative Mu Opiate Receptor gene expression was assessed by real time PCR during these seasons. The mRNA expression reached a relative peak during the month of June and was at its lowest in February and March, further demonstrating the direct coupling of morphine with this Receptor. We conclude that the temperature an animal is chronically exposed to serves to control cellular processes, i.e., Opiate signaling.
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identification of endogenous morphine and a Mu3 like Opiate alkaloid Receptor in human brain tissue taken from a patient with intractable complex partial epilepsy
Medical Science Monitor, 2008Co-Authors: Gregory L Fricchione, Patrick Cadet, Kirk J. Mantione, Wei Zhu, Edward B Bromfield, Joseph R Madsen, Umberto Degirolami, Barbara A Dworetzky, Bernardino Vaccaro, Peter Mcl BlackAbstract: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-polynierase chain reaction (RT-PCR) for the expression of different Mu Opiate Receptor gene transcripts. RIA revealed the presence of morphine at 3.4 nanograins 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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Differential expression of the human Mu Opiate Receptor from different primary vascular endothelial cells
Medical science monitor : international medical journal of experimental and clinical research, 2004Co-Authors: Patrick Cadet, Kirk J. Mantione, Thomas V. Bilfinger, George B. StefanoAbstract: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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Mu Opiate Receptor subtypes.
Medical science monitor : international medical journal of experimental and clinical research, 2004Co-Authors: Patrick CadetAbstract: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.
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Nitric oxide modulates the physiological control of ciliary activity in the marine Mussel Mytilus edulis via morphine: novel Mu Opiate Receptor splice variants.
Neuro endocrinology letters, 2004Co-Authors: Patrick CadetAbstract: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.
Abba J Kastin - One of the best experts on this subject based on the ideXlab platform.
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Endomorphins exit the brain by a saturable efflux system at the basolateral surface of cerebral endothelial cells
Experimental Brain Research, 2004Co-Authors: Aniko Somogyvari-vigh, James E Zadina, Abba J Kastin, Jie Liao, Weihong PanAbstract:Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two highly selective Mu-Opiate Receptor agonists. We recently demonstrated that EM-1 and EM-2 have a saturable transport system from brain-to-blood in vivo. Since the endothelial cells are the main component of the non-fenestrated microvessels of the blood-brain barrier (BBB), we examined whether these endogenous tetrapeptides have a saturable transport system in cultured cerebral endothelial cells. EM-1 and EM-2 binding and transport were studied in a transwell system in which primary mouse endothelial cells were co-cultured with rat glioma cells. We found that binding of both endomorphins was greater on the basolateral than the apical cell surface. Flux of EM-1 and EM-2 occurred predominantly in the basolateral to apical direction, each showing self-inhibition with an excess of the respective endomorphin. Transport was not influenced by the addition of the P-glycoprotein inhibitor, cyclosporin A. Neither the Mu-Opiate Receptor agonist DAMGO nor the delta-Opiate Receptor agonist DPDPE had any effect on the transport. Thus, the results show that a saturable transport system for EM-1 and EM-2 occurs at the level of endothelial cells of the BBB, and unlike ß-endorphin and morphine, P-glycoprotein is not needed for the brain-to-blood transport. Cross-inhibition of the transport of each endomorphin by the other suggests a shared transport system that is different from Mu- or delta-Opiate Receptors. As endormorphins are mainly produced in the CNS, the presence of the efflux system at the BBB could play an important role in pain modulation and neuroendocrine control.
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Facilitated Delivery of Endomorphins and Morphine into the CNS
2001Co-Authors: Abba J KastinAbstract:Abstract : Endomorphins, endogenous brain Opiates with the highest affinity and specifity for the Mu Opiate Receptor, potently produce analgesia. A rapid brain-to-blood efflux system could give misleading results when entry rates are determined. Preliminary results show that endomorphin-1 and endomorphin-2 are saturably transported from brain to blood, as shown by self-inhibition by an excess of that peptide. There also was cross-inhibition of each endomorphin by the other, indicating shared components for the efflux system. CGRP, substance P, or constriction of the sciatic nerve did not decrease efflux. Furthermore, chronic pain induced by sciatic nerve constriction caused a striking decrease in endomorphin-2 imMunoreactivity on the nerve-injured side in the spinal cord.
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Endomorphins: novel endogenous Mu-Opiate Receptor agonists in regions of high Mu-Opiate Receptor density.
Annals of the New York Academy of Sciences, 1999Co-Authors: James E Zadina, Abba J Kastin, Laszlo Hackler, S Martin-schild, Arnold A. Gerall, Xing ZhangAbstract:Abstract: Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM-1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-2) are peptides recently isolated from brain that show the highest affinity and selectivity for the m (morphine) Opiate Receptor of all the known endogenous opioids. The endomorphins have potent analgesic and gastrointestinal effects. At the cellular level, they activate G-proteins (35S-GTP γ-S binding) and inhibit calcium currents. Support for their role as endogenous ligands for the μ-Opiate Receptor includes their localization by radioimMunoassay and imMunocytochemistry in central nervous system regions of high μ Receptor density. Intense EM-2 imMunoreactivity is present in the terminal regions of primary afferent neurons in the dorsal horn of the spinal cord and in the medulla near high densities of μ Receptors. Chemical (capsaicin) and surgical (rhizotomy) disruption of nociceptive primary afferent neurons depletes the imMunoreactivity, implicating the primary afferents as the source of EM-2. Thus, EM-2 is well-positioned to serve as an endogenous modulator of pain in its earliest stages of perception. In contrast to EM-2, which is more prevalent in the spinal cord and lower brainstem, EM-1 is more widely and densely distributed throughout the brain than EM-2. The distribution is consistent with a role for the peptides in the modulation of diverse functions, including autonomic, neuroendocrine, and reward functions as well as modulation of responses to pain and stress.
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Endomorphin-2 is an endogenous opioid in primary sensory afferent fibers
Peptides, 1998Co-Authors: S Martin-schild, Abba J Kastin, Arnold A. Gerall, James E ZadinaAbstract:Evidence is presented that the recently discovered endogenous Mu-selective agonist, endomorphin-2, is localized in primary sensory afferents. Endomorphin-2-like imMunoreactivity was found to be colocalized in a subset of substance P- and Mu Opiate Receptor-containing fibers in the superficial laminae of the spinal cord and spinal trigeminal nucleus. Disruption of primary sensory afferents by mechanical (deafferentation by dorsal rhizotomy) or chemical (exposure to the primary afferent neurotoxin, capsaicin) methods virtually abolished endomorphin-2-like imMunoreactivity in the dorsal horn. These results indicate that endomorphin-2 is present in primary afferent fibers where it can serve as the endogenous ligand for pre- and postsynaptic Mu Receptors and as a major modulator of pain perception.
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a potent and selective endogenous agonist for the Mu Opiate Receptor
Nature, 1997Co-Authors: James E Zadina, Laszlo Hackler, Linjun Ge, Abba J KastinAbstract:Peptides have been identified in mammalian brain that are considered to be endogenous agonists for the δ (enkephalins) and κ (dynorphins) Opiate Receptors, but none has been found to have any preference for the µ Receptor1–3. Because morphine and other compounds that are clinically useful and open to abuse act primarily at the µ Receptor4, it could be important to identify endogenous peptides specific for this site. Here we report the discovery and isolation from brain of such a peptide, endomorphin-1 (Tyr-Pro-Trp-Phe-NH2), which has a high affinity (Ki = 360 pM) and selectivity (4,000- and 15,000-fold preference over the δ and κ Receptors) for the µ, Receptor. This peptide is more effective than the µ-selective analogue DAMGO in vitroand it produces potent and prolonged analgesia in mice. A second peptide, endomorphin-2 (Tyr-Pro-Phe-Phe-NH2), which differs by one amino acid, was also isolated. The new peptides have the highest specificity and affinity for the µ Receptor of any endogenous substance so far described and they maybe natural ligands for this Receptor.
Donna E Webster - One of the best experts on this subject based on the ideXlab platform.
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black cohosh actaea racemosa cimicifuga racemosa behaves as a mixed competitive ligand and partial agonist at the human Mu Opiate Receptor
Journal of Agricultural and Food Chemistry, 2006Co-Authors: Meera Rhyu, Donna E Webster, Norman R Farnsworth, Daniel S Fabricant, Jim Z WangAbstract:Black cohosh is a commonly used botanical dietary supplement for the treatment of climacteric complaints. Because the Opiate system in the brain is intimately associated with mood, temperature, and sex hormonal levels, the activity of black cohosh extracts at the human μ Opiate Receptor (hMOR) expressed in Chinese hamster ovary cells was investigated. The 100% methanol, 75% ethanol, and 40% 2-propanol extracts of black cohosh effectively displaced the specific binding of [3H]DAMGO to hMOR. Further studies of the clinically used ethanol extract indicated that black cohosh acted as a mixed competitive ligand, displacing 77 ± 4% [3H]DAMGO to hMOR (Ki = 62.9 μg/mL). Using the [35S]GTPγS assay, the action of black cohosh was found to be consistent with an agonist, with an EC50 of 68.8 ± 7.7 μg/mL. These results demonstrate for the first time that black cohosh contains active principle(s) that activate hMOR, supporting its beneficial role in alleviating menopausal symptoms. Keywords: Black cohosh; menopause; ho...
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activation of the μ Opiate Receptor by vitex agnus castus methanol extracts implication for its use in pms
Journal of Ethnopharmacology, 2006Co-Authors: Donna E Webster, Shaonong Chen, Norman R Farnsworth, Jim Z WangAbstract:The dried ripe fruit of Vitex agnus-castus L. (VAC) is widely used for the treatment of premenstrual syndrome (PMS). A previous study reported that extracts of VAC showed affinity to Opiate Receptors; however, functional activity was not determined. We tested two different VAC extracts in Receptor binding and functional assays. Our objectives were: (1) to confirm the Opiate affinity; (2) to rule out interference by free fatty acids (FFA); (3) to determine the mode of action of VAC at the Mu-Opiate Receptor. Methanol extracts of VAC were prepared either before (VAC-M1) or after (VAC-M2) extraction with petroleum ether to remove fatty acids. Both extracts showed significant affinities to the Mu-Opiate Receptor, as indicated by the concentration-dependent displacement of [3H]DAMGO binding in Chinese hamster ovary (CHO)-human Mu-Opiate Receptor (hMOR) cells. The IC50 values were estimated to be 159.8 microg/ml (VAC-M1) and 69.5 microg/ml (VAC-M2). Since the defatted extract not only retained, but exhibited a higher affinity (p<0.001), it argued against significant interference by fatty acids. In an assay to determine Receptor activation, VAC-M1 and VAC-M2 stiMulated [35S]GTPgammaS binding by 41 and 61% (p<0.001), respectively. These results suggested for the first time that VAC acted as an agonist at the Mu-Opiate Receptor, supporting its beneficial action in PMS.
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Activation of the μ-Opiate Receptor by Vitex agnus-castus methanol extracts : Implication for its use in PMS
Journal of ethnopharmacology, 2006Co-Authors: Donna E Webster, Shaonong Chen, Norman R Farnsworth, Z. Jim WangAbstract:The dried ripe fruit of Vitex agnus-castus L. (VAC) is widely used for the treatment of premenstrual syndrome (PMS). A previous study reported that extracts of VAC showed affinity to Opiate Receptors; however, functional activity was not determined. We tested two different VAC extracts in Receptor binding and functional assays. Our objectives were: (1) to confirm the Opiate affinity; (2) to rule out interference by free fatty acids (FFA); (3) to determine the mode of action of VAC at the Mu-Opiate Receptor. Methanol extracts of VAC were prepared either before (VAC-M1) or after (VAC-M2) extraction with petroleum ether to remove fatty acids. Both extracts showed significant affinities to the Mu-Opiate Receptor, as indicated by the concentration-dependent displacement of [3H]DAMGO binding in Chinese hamster ovary (CHO)-human Mu-Opiate Receptor (hMOR) cells. The IC50 values were estimated to be 159.8 microg/ml (VAC-M1) and 69.5 microg/ml (VAC-M2). Since the defatted extract not only retained, but exhibited a higher affinity (p
James E Zadina - One of the best experts on this subject based on the ideXlab platform.
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Endomorphins exit the brain by a saturable efflux system at the basolateral surface of cerebral endothelial cells
Experimental Brain Research, 2004Co-Authors: Aniko Somogyvari-vigh, James E Zadina, Abba J Kastin, Jie Liao, Weihong PanAbstract:Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two highly selective Mu-Opiate Receptor agonists. We recently demonstrated that EM-1 and EM-2 have a saturable transport system from brain-to-blood in vivo. Since the endothelial cells are the main component of the non-fenestrated microvessels of the blood-brain barrier (BBB), we examined whether these endogenous tetrapeptides have a saturable transport system in cultured cerebral endothelial cells. EM-1 and EM-2 binding and transport were studied in a transwell system in which primary mouse endothelial cells were co-cultured with rat glioma cells. We found that binding of both endomorphins was greater on the basolateral than the apical cell surface. Flux of EM-1 and EM-2 occurred predominantly in the basolateral to apical direction, each showing self-inhibition with an excess of the respective endomorphin. Transport was not influenced by the addition of the P-glycoprotein inhibitor, cyclosporin A. Neither the Mu-Opiate Receptor agonist DAMGO nor the delta-Opiate Receptor agonist DPDPE had any effect on the transport. Thus, the results show that a saturable transport system for EM-1 and EM-2 occurs at the level of endothelial cells of the BBB, and unlike ß-endorphin and morphine, P-glycoprotein is not needed for the brain-to-blood transport. Cross-inhibition of the transport of each endomorphin by the other suggests a shared transport system that is different from Mu- or delta-Opiate Receptors. As endormorphins are mainly produced in the CNS, the presence of the efflux system at the BBB could play an important role in pain modulation and neuroendocrine control.
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The effects of endomorphin-1 on conditioned defeat in Syrian hamsters (Mesocricetus auratus).
Brain research, 2001Co-Authors: R D Whitten, James E Zadina, A M Jasnow, H E Albers, S Martin-schild, K L HuhmanAbstract:The present study examined the effect of endomorphin-1 (EM1), an endogenous opioid with a high affinity for the Mu Opiate Receptor, on conditioned defeat. Conditioned defeat is a phenomenon in which hamsters that have been defeated subsequently fail to exhibit normal territorial aggression and instead display submissive/defensive behaviors even when paired with a non-aggressive intruder. In experiment 1, animals were placed in the home cage of a larger resident for 15 min and were defeated. After 24 h, animals received a 3-microl injection of EM1 (0.0, 0.3, 3.0, or 10 nmol) into the left lateral cerebral ventricle 5 min before a smaller non-aggressive intruder was placed in the home cage of the experimental animal. In experiment 2, animals were infused with EM1 immediately after the initial defeat and were paired with a non-aggressive intruder 24 h later as in experiment 1. EM1 reduced the duration of submissive/defensive behavior in experiment 1 (P0.05). These data support the hypothesis that the highly selective Mu Receptor agonist endomorphin-1 modulates the expression of conditioned defeat, but provides no support for the hypothesis that endomorphin-1 modulates the consolidation of conditioned defeat.
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Endomorphins: novel endogenous Mu-Opiate Receptor agonists in regions of high Mu-Opiate Receptor density.
Annals of the New York Academy of Sciences, 1999Co-Authors: James E Zadina, Abba J Kastin, Laszlo Hackler, S Martin-schild, Arnold A. Gerall, Xing ZhangAbstract:Abstract: Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM-1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-2) are peptides recently isolated from brain that show the highest affinity and selectivity for the m (morphine) Opiate Receptor of all the known endogenous opioids. The endomorphins have potent analgesic and gastrointestinal effects. At the cellular level, they activate G-proteins (35S-GTP γ-S binding) and inhibit calcium currents. Support for their role as endogenous ligands for the μ-Opiate Receptor includes their localization by radioimMunoassay and imMunocytochemistry in central nervous system regions of high μ Receptor density. Intense EM-2 imMunoreactivity is present in the terminal regions of primary afferent neurons in the dorsal horn of the spinal cord and in the medulla near high densities of μ Receptors. Chemical (capsaicin) and surgical (rhizotomy) disruption of nociceptive primary afferent neurons depletes the imMunoreactivity, implicating the primary afferents as the source of EM-2. Thus, EM-2 is well-positioned to serve as an endogenous modulator of pain in its earliest stages of perception. In contrast to EM-2, which is more prevalent in the spinal cord and lower brainstem, EM-1 is more widely and densely distributed throughout the brain than EM-2. The distribution is consistent with a role for the peptides in the modulation of diverse functions, including autonomic, neuroendocrine, and reward functions as well as modulation of responses to pain and stress.
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Endomorphin-2 is an endogenous opioid in primary sensory afferent fibers
Peptides, 1998Co-Authors: S Martin-schild, Abba J Kastin, Arnold A. Gerall, James E ZadinaAbstract:Evidence is presented that the recently discovered endogenous Mu-selective agonist, endomorphin-2, is localized in primary sensory afferents. Endomorphin-2-like imMunoreactivity was found to be colocalized in a subset of substance P- and Mu Opiate Receptor-containing fibers in the superficial laminae of the spinal cord and spinal trigeminal nucleus. Disruption of primary sensory afferents by mechanical (deafferentation by dorsal rhizotomy) or chemical (exposure to the primary afferent neurotoxin, capsaicin) methods virtually abolished endomorphin-2-like imMunoreactivity in the dorsal horn. These results indicate that endomorphin-2 is present in primary afferent fibers where it can serve as the endogenous ligand for pre- and postsynaptic Mu Receptors and as a major modulator of pain perception.
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a potent and selective endogenous agonist for the Mu Opiate Receptor
Nature, 1997Co-Authors: James E Zadina, Laszlo Hackler, Linjun Ge, Abba J KastinAbstract:Peptides have been identified in mammalian brain that are considered to be endogenous agonists for the δ (enkephalins) and κ (dynorphins) Opiate Receptors, but none has been found to have any preference for the µ Receptor1–3. Because morphine and other compounds that are clinically useful and open to abuse act primarily at the µ Receptor4, it could be important to identify endogenous peptides specific for this site. Here we report the discovery and isolation from brain of such a peptide, endomorphin-1 (Tyr-Pro-Trp-Phe-NH2), which has a high affinity (Ki = 360 pM) and selectivity (4,000- and 15,000-fold preference over the δ and κ Receptors) for the µ, Receptor. This peptide is more effective than the µ-selective analogue DAMGO in vitroand it produces potent and prolonged analgesia in mice. A second peptide, endomorphin-2 (Tyr-Pro-Phe-Phe-NH2), which differs by one amino acid, was also isolated. The new peptides have the highest specificity and affinity for the µ Receptor of any endogenous substance so far described and they maybe natural ligands for this Receptor.