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Unfolding of acetylcholinesterase from Bungarus fasciatus
S46 LONG-TERM INTERACTIONS BETWEEN OPIOID AND CANNABINOID DRUGS IN N18TG2 NEUROBLASTOMA CELLS M. Shapira, M. Gafni and Y. Sarne Dept. of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
BLOCKING CCK-A RECEPTORS IN NEONATAL RAT PUPS INCREASES THEIR NATURAL PREFERENCES TOWARDS STIMULI REPRESENTING THE NEST M. Shayit and A. Weller Developmental Psychobiology Lab., Dept. of Psychology, Bar-Ilan University, Ramat Gan, Israel
Cannabinoid and opioid agonists represent two separate groups of psychoactive drugs that share a similar pharmacological profile. Cross tolerance between opioids and cannabinoids has been previously found in various in-vivo studies. In the present study, we explored long-term interactions between opioids and cannabinoids at the cellular level. For this purpose, we used N18TG2 neuroblastoma cells that co-express both d-opioid and CB1-cannabinoid receptors. G-protein activation was measured by the binding of [35S]GTPgS. [35S]GTPgS binding to N18TG2 membranes was stimulated by either opioid or cannabinoid agonists. Chronic treatment of the cells with opioid or with cannabinoid agonists induced desensitization to the respective drug. Asymmetric cross tolerance was found between these two groups of drugs: long-term exposure to the cannabinoid agonist deacetyllevonantradol (DALN) induced homologous desensitization and did not reduce the effect of opioids, while long-term exposure to the opioid agonist etorphine induced heterologous desensitization and partially reduced the cannabinoid effect on [35S]GTPgS binding. Chronic exposure of cell cultures to DALN or to etorphine not only induced desensitization, but also elevated the basal activity of G-proteins in the exposed cells. The combination of the two drugs did not yield an additive elevation, suggesting that chronic exposure of the cells to cannabinoid and opioid agonists modified a common responding element within N18TG2 cells. This work presents the N18TG2 neuroblastoma cell line as a suitable experimental model to study the molecular mechanism(s) underlying chronic interactions between opioid and cannabinoid drugs.
Cholecystokinin (CCK), a gut hormone and neuropeptide, produces various physiological and behavioral effects. We examined the role of the CCK system of rat pups in the expression of natural preferences, towards tactile, olfactory and temperature stimuli, chosen to represent important aspects of the mother and nest. Devazepide (600 or 1000 Pg/kg), a potent and selective CCK-A receptor antagonist, was administered IP to 9–12 day old rats, and a preference test was performed in a test box. In Exp. 1, one side contained the odor of maternal feces, the other remained empty. In Exp. 2, soft carpet covered one side of the floor, the other was covered by plywood. In Exp. 3, floor temperature was 31°C–32°C, or about 25°C. Relative preference was measured by the time spent on each side of the test box, for six 30-s trials (Exps. 1 and 2), and four 90-s trials (Exp. 3). The results did not show an overall difference in mean time spent near the odor. Still, a significant interaction was obtained: time spent near the odor in the experimental groups increased over time, while it decreased in the control group. Similarly in Exp. 2, a significant interaction, indicated an increment in preference rate of the Devazepide (600 Pg/kg) group, over time, compared to a decrement in the control group. In Exp. 3, subjects receiving Devazepide 600 Pg/kg, spent significantly more time over the warmer side, over all, compared to controls. These results support the hypothesis that blocking CCK-A receptors, may increase the preference rate for stimuli representing important aspects of the nest and dam.
EFFECTS OF PRENATAL MORPHINE EXPOSURE ON BEHAVIORAL RESPONSES TO LPS IN RATS. Y. Shavit, E. Cohen and R. Yirmiya Dept. of Psychology, The Hebrew University of Jerusalem, Jerusalem, Israel Prenatal exposure to opiates can adversely affect fetal development, resulting in long-term growth retardation and impairments in physiological and behavioral functions. In the present study we studied longterm effects of prenatal morphine exposure on immune functions, including the activity of natural killer (NK) cells and the febrile and behavioral responses to lipopolysaccharide (LPS). Pregnant Fischer 344 rats were given increasing doses of morphine in slow release emulsion during gestational days 12–18. Control rats were injected with vehicle and were either pair-fed to morphine rats or fed ad-libitum. Postnatal experiments were conducted when offspring were 10–12 weeks old. Compared to both control groups, rats prenatally exposed to morphine exhibited: (1) Suppressed cytotoxic activity of NK cells; (2) Reduced LPS-induced fever measured by a biotelemetric system; (3) Reduced hyperalgesia measured by the hot-plate test at 30 min, and augmented hypoalgesia at 2–6 h post-LPS; (4) Higher open field activity in salinetreated animals, and more pronounced suppression of activity in LPSinjected animals; (5) LPS-induced reduction of food consumption, body weight, and social exploration, which did not differ from the reduction observed in control animals. These findings indicate that prenatal exposure to morphine induces long term impairment of host-defense mechanisms, including NK activity and responsiveness to endotoxin challenge. Prenatal morphine rats exhibited suppressed NK activity, reduced febrile response and impaired regulation of LPS-induced sickness behavior, suggesting that exposure to opiates during pregnancy renders the offspring more susceptible to infectious diseases. Supported by a grant from the Israel Foundation Trustees (Y.S.).
UNFOLDING OF ACETYLCHOLINESTERASE FROM BUNGARUS FASCIATUS I. Shin*, I. Silman*, C. Bon‡, and L. Weiner† Depts. of Neurobiology* and Organic Chemistry†, Weizmann Institute of Science, Rehovot, Israel;. ‡Unité des Venins, Institut Pasteur, Paris, France The possible effect of unilamellar liposomes composed of dimyristoylphosphatidylcholine (DMPC) on unfolding of acetylcholinesterase from Bungarus fasciatus venom (BfAChE), a monomeric water-soluble enzyme, was investigated. The kinetics of the thermal inactivation of BfAChE were studied in the temperature range of 45–54°C. An Arrhenius plot revealed an activation energy of 113 kcal/mol. Thermally denatured BfAChE displays spectroscopic characteristics typical of a partially unfolded ‘molten globule’ (MG) state. The rate of thermal denaturation was greatly enhanced in the presence of unilamellar DMPC vesicles. An Arrhenius plot revealed that the energy barrier for transition from the native to the thermally denatured state is lowered from 113 to 52 kcal/mol in the presence of the liposomes. It was found that, in contrast to the MG of Torpedo AChE (Shin et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2848–2852), the thermally denatured BfAChE does not remain bound to the liposomes, but is released after unfolding, and subsequently aggregates. A catalytic scheme for liposome-mediated thermal denaturation was proposed by us. It was shown that the rate of unfolding of BfAChE in the presence of liposomes can be described by the Michaelis-Menten equation (Km = 2.10–6 M). DMPC vesicles display a catalytic turnover number of kcat ~100 min−1, assuming one binding site per vesicle for BfAChE. Our findings raise the possibility that the membrane itself, by lowering the energy barrier for transition to a partially unfolded state, may play an active role in insertion and translocation of proteins in situ.
STUDY OF THE ASSOCIATION BETWEEN D6S461 AND MULTIPLE SCLEROSIS IN JEWISH PATIENTS
BLOCKING CCK-A RECEPTORS IN NEONATAL RAT PUPS INCREASES THEIR NATURAL PREFERENCES TOWARDS STIMULI REPRESENTING THE NEST M. Shayit and A. Weller Developmental Psychobiology Lab., Dept. of Psychology, Bar-Ilan University, Ramat Gan, Israel
Cannabinoid and opioid agonists represent two separate groups of psychoactive drugs that share a similar pharmacological profile. Cross tolerance between opioids and cannabinoids has been previously found in various in-vivo studies. In the present study, we explored long-term interactions between opioids and cannabinoids at the cellular level. For this purpose, we used N18TG2 neuroblastoma cells that co-express both d-opioid and CB1-cannabinoid receptors. G-protein activation was measured by the binding of [35S]GTPgS. [35S]GTPgS binding to N18TG2 membranes was stimulated by either opioid or cannabinoid agonists. Chronic treatment of the cells with opioid or with cannabinoid agonists induced desensitization to the respective drug. Asymmetric cross tolerance was found between these two groups of drugs: long-term exposure to the cannabinoid agonist deacetyllevonantradol (DALN) induced homologous desensitization and did not reduce the effect of opioids, while long-term exposure to the opioid agonist etorphine induced heterologous desensitization and partially reduced the cannabinoid effect on [35S]GTPgS binding. Chronic exposure of cell cultures to DALN or to etorphine not only induced desensitization, but also elevated the basal activity of G-proteins in the exposed cells. The combination of the two drugs did not yield an additive elevation, suggesting that chronic exposure of the cells to cannabinoid and opioid agonists modified a common responding element within N18TG2 cells. This work presents the N18TG2 neuroblastoma cell line as a suitable experimental model to study the molecular mechanism(s) underlying chronic interactions between opioid and cannabinoid drugs.
Cholecystokinin (CCK), a gut hormone and neuropeptide, produces various physiological and behavioral effects. We examined the role of the CCK system of rat pups in the expression of natural preferences, towards tactile, olfactory and temperature stimuli, chosen to represent important aspects of the mother and nest. Devazepide (600 or 1000 Pg/kg), a potent and selective CCK-A receptor antagonist, was administered IP to 9–12 day old rats, and a preference test was performed in a test box. In Exp. 1, one side contained the odor of maternal feces, the other remained empty. In Exp. 2, soft carpet covered one side of the floor, the other was covered by plywood. In Exp. 3, floor temperature was 31°C–32°C, or about 25°C. Relative preference was measured by the time spent on each side of the test box, for six 30-s trials (Exps. 1 and 2), and four 90-s trials (Exp. 3). The results did not show an overall difference in mean time spent near the odor. Still, a significant interaction was obtained: time spent near the odor in the experimental groups increased over time, while it decreased in the control group. Similarly in Exp. 2, a significant interaction, indicated an increment in preference rate of the Devazepide (600 Pg/kg) group, over time, compared to a decrement in the control group. In Exp. 3, subjects receiving Devazepide 600 Pg/kg, spent significantly more time over the warmer side, over all, compared to controls. These results support the hypothesis that blocking CCK-A receptors, may increase the preference rate for stimuli representing important aspects of the nest and dam.
EFFECTS OF PRENATAL MORPHINE EXPOSURE ON BEHAVIORAL RESPONSES TO LPS IN RATS. Y. Shavit, E. Cohen and R. Yirmiya Dept. of Psychology, The Hebrew University of Jerusalem, Jerusalem, Israel Prenatal exposure to opiates can adversely affect fetal development, resulting in long-term growth retardation and impairments in physiological and behavioral functions. In the present study we studied longterm effects of prenatal morphine exposure on immune functions, including the activity of natural killer (NK) cells and the febrile and behavioral responses to lipopolysaccharide (LPS). Pregnant Fischer 344 rats were given increasing doses of morphine in slow release emulsion during gestational days 12–18. Control rats were injected with vehicle and were either pair-fed to morphine rats or fed ad-libitum. Postnatal experiments were conducted when offspring were 10–12 weeks old. Compared to both control groups, rats prenatally exposed to morphine exhibited: (1) Suppressed cytotoxic activity of NK cells; (2) Reduced LPS-induced fever measured by a biotelemetric system; (3) Reduced hyperalgesia measured by the hot-plate test at 30 min, and augmented hypoalgesia at 2–6 h post-LPS; (4) Higher open field activity in salinetreated animals, and more pronounced suppression of activity in LPSinjected animals; (5) LPS-induced reduction of food consumption, body weight, and social exploration, which did not differ from the reduction observed in control animals. These findings indicate that prenatal exposure to morphine induces long term impairment of host-defense mechanisms, including NK activity and responsiveness to endotoxin challenge. Prenatal morphine rats exhibited suppressed NK activity, reduced febrile response and impaired regulation of LPS-induced sickness behavior, suggesting that exposure to opiates during pregnancy renders the offspring more susceptible to infectious diseases. Supported by a grant from the Israel Foundation Trustees (Y.S.).
UNFOLDING OF ACETYLCHOLINESTERASE FROM BUNGARUS FASCIATUS I. Shin*, I. Silman*, C. Bon‡, and L. Weiner† Depts. of Neurobiology* and Organic Chemistry†, Weizmann Institute of Science, Rehovot, Israel;. ‡Unité des Venins, Institut Pasteur, Paris, France The possible effect of unilamellar liposomes composed of dimyristoylphosphatidylcholine (DMPC) on unfolding of acetylcholinesterase from Bungarus fasciatus venom (BfAChE), a monomeric water-soluble enzyme, was investigated. The kinetics of the thermal inactivation of BfAChE were studied in the temperature range of 45–54°C. An Arrhenius plot revealed an activation energy of 113 kcal/mol. Thermally denatured BfAChE displays spectroscopic characteristics typical of a partially unfolded ‘molten globule’ (MG) state. The rate of thermal denaturation was greatly enhanced in the presence of unilamellar DMPC vesicles. An Arrhenius plot revealed that the energy barrier for transition from the native to the thermally denatured state is lowered from 113 to 52 kcal/mol in the presence of the liposomes. It was found that, in contrast to the MG of Torpedo AChE (Shin et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2848–2852), the thermally denatured BfAChE does not remain bound to the liposomes, but is released after unfolding, and subsequently aggregates. A catalytic scheme for liposome-mediated thermal denaturation was proposed by us. It was shown that the rate of unfolding of BfAChE in the presence of liposomes can be described by the Michaelis-Menten equation (Km = 2.10–6 M). DMPC vesicles display a catalytic turnover number of kcat ~100 min−1, assuming one binding site per vesicle for BfAChE. Our findings raise the possibility that the membrane itself, by lowering the energy barrier for transition to a partially unfolded state, may play an active role in insertion and translocation of proteins in situ.
STUDY OF THE ASSOCIATION BETWEEN D6S461 AND MULTIPLE SCLEROSIS IN JEWISH PATIENTS