Weizmann Logo Neurobiology Department
Weizmann Institute of Science
Prof. Zvi Vogel's laboratory

List of Recent Publications (1995 - 2001)

99. Blechman J.M., Lev S., Barg J., Eisenstein M., Vaks B., Vogel Z., Givol D. and Yarden Y. (1995) The 4th immunoglobulin domain of the stem-cell factor-receptor couples ligand-binding to signal-transduction. Cell 80:103-113.

Abstract: Receptor dimerization is ubiquitous to the action of all receptor tyrosine kinases, and in the case of dimeric ligands, such as the stem cell factor (SCF), it was attributed to ligand bivalency. However, by using a dimerization-inhibitory monoclonal antibody to the SCF receptor, we confined a putative dimerization site to the nonstandard fourth immunoglobulin-like domain of the receptor. Deletion of this domain not only abolished ligand-induced dimerization and completely inhibited signal transduction, but also provided insights into the mechanism of the coupling of ligand binding to dimer formation. These results identify an intrinsic receptor dimerization site and suggest that similar sites may exist in other receptors.

100. Fride E., Barg J., Levy R., Saya D., Heldman E., Mechoulam R. and Vogel Z. (1995) Low doses of anandamides inhibit pharmacological effects of [delta]9-tetrahydrocannabinol. J. Pharmacol. Exp. Ther. 272:699-707.

Abstract: It has been shown previously that the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide 20:4, n-6) induces in vivo and in vitro effects typical of a cannabinoid partial agonist. We now report that the synthetic docosahexaenytethanolamide (anandamide 22:6, n-3) shows similar activities. In addition we show that these two anandamides, under certain experimental conditions, antagonize the effects of Delta(9)-THC both in vivo and in vitro. Thus a significant decrease in the potency of Delta(9)-THC-induced inhibition of adenylate cyclase was observed in N18TG2 neuroblastoma cells that were pretreated with low concentrations of anandamides. At these low concentrations of anandamides had no effect when applied alone. In vivo, Sabra or ICR mice were subjected to a tetrad of tests, designed to detect cannabinoid-induced effects. Mice pretreated (i.p.) with 10 mg/kg of Delta(9)-THC received injections with anandamides. Only low doses (0.0001- 0.1 mg/kg) of the anandamides, which had no effects when administered alone, partially or fully inhibited the THC- induced effects. These findings suggest that the inhibition of Delta(9)-THC-induced effects by low doses of anandamides may be due to partial agonistic effects of these materials. It is possible that low doses of the anandamides are capable of activating a Gs protein mediated signaling pathway, or may cause an allosteric modulation of the cannabinoid receptor.

101. Mechoulam R., Ben-Shabat S., Hanus L., Ligumsky M., Kaminski N.E., Schatz A.R., Gopher A., Almog S., Martin B.R., Compton D.R., Pertwee R.G., Griffin G., Bayewitch M., Barg J. and Vogel Z. (1995) Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem. Pharmacol. 50:83-90.
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Abstract: In this study, we report the isolation from canine intestines of 2-arachidonyl glycerol (2-Ara-Gl). Its structure was determined by mass spectrometry and by direct comparison with a synthetic sample. 2-Ara-Gl bound to membranes from cells transiently transfected with expression plasmids carrying DNA of either CB1 or CB2-the two cannabinoid receptors identified thus far-with K-i values of 472 +/- 55 and 1400 +/- 172 nM, respectively. In the presence of forskolin, 2-Ara-Gl inhibited adenylate cyclase in isolated mouse spleen cells, at the potency level of Delta(9)-tetrahydrocannabinol (Delta(9)-THC). Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature. 2-Ara-Gl also shares the ability of Delta(9)-THC to inhibit electrically evoked contractions of mouse isolated vasa deferentia; however, it was less potent than Delta(9)-THC.

102. Avidor-Reiss T., Zippel R., Levy R., Saya D., Ezra V., Barg J., Matus-Leibovitch N. and Vogel Z. (1995) [kappa]-Opioid receptor-transfected cell lines - Modulation of adenylyl-cyclase activity following acute and chronic opioid treatments. FEBS Lett. 361:70-74.
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Abstract: The opioid receptors mu, delta and kappa have recently been cloned. Here me show that kappa-agonists inhibit adenylyl cyclase activity in Chinese hamster ovary cells stably transfected with rat kappa-opioid receptor cDNA. Chronic exposure of the cells to kappa-agonists did not lead to significant desensitization of the capacity of the agonists to inhibit adenylyl cyclase. On the other hand, withdrawal of the agonist following the chronic treatment led to the phenomenon of supersensitivity ('overshoot') of adenylyl cyclase activity. Both the inhibition of adenylyl cyclase activity by the acute opioid treatment and the chronic agonist-induced supersensitivity are pertussis toxin sensitive, demonstrating involvement of G(i)/G(o) proteins in both processes.

103. Barg J., Fride E., Hanus L., Levy R., Matus-Leibovitch N., Heldman E., Bayewitch M., Mechoulam R. and Vogel Z. (1995) Cannabinomimetic behavioral effects of and adenylate cyclase inhibition by two new endogenous anandamides. Eur. J. Pharmacol. 287:145-152.
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Abstract: We have previously shown that the endogenous putative cannabinoid ligand arachidonylethanolamide (anandamide, 20:4, n - 6) induces in vivo and in vitro effects typical of a cannabinoid agonist. We now report that two other endogenous anandamides, docosatetraenylethanoiamide (anandamide, 22:4, n - 6) and homo-gamma-linolenylethanolamide (anandamide, 20:3, n - 6), have similar activities. The new anandamides bind to SV40- transformed African green monkey kidney cells transfected with the rat brain cannabinoid receptor cDNA and display K-I values of 253.4 +/- 41.1 and 244.8 +/- 38.7, respectively. The value found for arachidonylethanolamide was 155.1 +/- 13.8 nM. In addition, the new anandamides inhibit prostaglandin E(1)- stimulated adenylate cyclase activity in Chinese hamster ovary- K-1 cells transfected with the cannabinoid receptor, as well as in N(18)TG(2) mouse neuroblastoma cells that express the cannabinoid receptor naturally. The IC,, values for the inhibition of adenylate cyclase in transfected Chinese hamster ovary-K-1 cells were 116.8 +/- 8.7 and 109.3 +/- 8.6 nM for docosatetraenylethanolamide and homo-gamma- linolenylethanolamide, respectively. These values were similar to that obtained with arachidonylethanolamide (100.5 +/- 7.7 nM), but were significantly higher than the IC50 value observed with the plant cannabinoid Delta(9)-tetrahydrocannabinol (9.2 +/- 8.6 nM). The inhibitory effects of the anandamides on adenylate cyclase activity were blocked by pertussis toxin, indicating the involvement of pertussis toxin-sensitive GTP- binding protein(s). In a tetrad of behavioral assays for cannabinoid-like effects, the two new anandamides exerted similar behavioral effects to those observed with Delta(9)- tetrahydrocannabinol and arachidonylethanolamide: inhibition of motor activity in an open field, hypothermia, catalepsy on a ring, and analgesia on a hot plate.

104. Matus-Leibovitch N., Ezra-Macabee V., Saya D., Attali B., Avidor-Reiss T., Barg J. and Vogel Z. (1995) Increased expression of synapsin I mRNA in defined areas of the rat central nervous system following chronic morphine treatment. Mol. Brain Res. 34:221-230.
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Abstract: Chronic opiate administration leads to a selective regulation of several cellular proteins and mRNAs. This phenomenon has been viewed as a compensatory mechanism to the opiate signaling leading to the development of opiate addiction. In this study, in situ hybridization histochemistry experiments were employed to investigate the effect of chronic morphine treatment on synapsin I gene expression. We show here for the first time that prolonged morphine exposure causes a selective increase in the mRNA levels of synapsin I in several brain regions which are considered to be important for opiate action. Quantitative analysis of the signals, obtained by hybridization of digoxigenin-labeled antisense RNA probe, revealed a 5.8- and 7- fold increase of synapsin I mRNA levels in the locus coeruleus and the amygdala of morphine-treated rats, respectively, as compared with control untreated rats. Increased expression of synapsin I mRNA was also observed in the spinal cord of morphine-treated rats (by 3.8-fold). Since opiates were shown to attenuate neurotransmitter release and reduce synapsin I phosphorylation, it is suggested that the increase in synapsin I levels would lead to the requirement of higher amounts of opiate agonists to obtain the opiate physiological effects. These results suggest that the increases in mRNA levels of synapsin I in these specific areas can be part of the molecular mechanism(s) underlying opiate tolerance and withdrawal.

105. Avidor-Reiss T., Bayewitch M., Levy R., Matus-Leibovitch N., Nevo I. and Vogel Z. (1995) Adenylylcyclase supersensitization in [mu]-opioid receptor-transfected Chinese hamster ovary cells following chronic opioid treatment. J. Biol. Chem. 270:29732-29738.
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Abstract: Using CHO cells stably transfected with rat mu-opioid receptor cDNA, we show that the mu-agonists morphine and [D-Ala(2),N- methyl-Phe(4),Gly-ol(5)]enkephalin are negatively coupled to adenylylcyclase and inhibit forskolin-stimulated cAMP accumulation. Chronic exposure of cells to morphine leads to the rapid development of tolerance. Withdrawal of morphine or [D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin following chronic treatment (by wash or addition of the antagonist naloxone) leads to an immediate increase in cyclase activity (supersensitization or overshoot), which is gradually reversed upon further incubation with naloxone. Phosphodiesterase inhibitors do not affect the overshoot, indicating that it results from cyclase stimulation rather than phosphodiesterase regulation, Morphine's potency to inhibit cAMP accumulation is the same before and after chronic treatment, suggesting that the apparent tolerance results from cyclase activation, rather than from receptor desensitization. The similar kinetics of induction of tolerance and overshoot support this idea. Both the overshoot and acute opioid-induced cyclase inhibition are blocked by naloxone and are pertussis toxin-sensitive, indicating that both phenomena are mediated by the mu-receptor and G(i)/G(o) proteins. The supersensitization is cycloheximide-insensitive, indicating that it does not require newly synthesized proteins. This is supported by the rapid development of supersensitization. Taken together, these results show that mu-transfected cells can serve as a model for investigating molecular and cellular mechanisms underlying opiate drug addiction.

106. Gorodinsky A., Barg J., Belcheva M.M., Levy R., McHale R.J., Vogel Z. and Coscia C.J. (1995) Dynorphins modulate DNA-synthesis in fetal brain-cell aggregates. J. Neurochem. 65:1481-1486.

Abstract: Previously, opioid peptide analogues, beta-endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7-day fetal rat brain cell aggregates via kappa-and mu-opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 [mu]M, all dynorphins tested and beta-endorphin inhibited [H-3]thymidine incorporation into DNA by 20-38% in 7-day rat brain cell aggregates. The putative epsilon-antagonist beta-endorphin (1- 27) did not prevent the effect of beta-endorphin, suggesting that the epsilon-receptor is not involved in opioid inhibition of DNA synthesis. The kappa-selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a kappa-opioid receptor. In dose- dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete kappa-receptor subtype, The IC50 value of 0.1 nM estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the kappa-receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21-day aggregates were treated with dynorphins, a 33-86% enhancement of thymidine incorporation was observed. Because both 7- and 21-day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express kappa-receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.

107. Novakova M., Ela C., Barg J., Vogel Z., Hasin Y. and Eilam Y. (1995) Inotropic action of sigma-receptor ligands in isolated cardiac myocytes from adult rats. Eur. J. Pharmacol. 286:19-30.
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Abstract: High affinity binding sites for a receptor ligands were found in membranes of cardiac myocytes from adult rats. The sigma receptor ligand (+)-3-hydroxyphenyl-N-(1-propyl)piperidine ((+)-3-PPP) binds with a K-d of 17.9 +/- 4.0 nM and a B-max of 275 +/- 32.1 fmol/mg protein. Competition experiments of (+)- pentazocine with [H-3]1,3-di-O-tolylguanidine ([H-3]DTG) binding yielded a K-i of 6.1 +/- 1.3 nM. The majority of the sites (> 80%) were of the sigma(1) subtype. Exposure of isolated cardiomyocytes from adult rats to (+)-3-PPP (10 nM-1.0 [mu]M) caused a marked concentration-dependent increase in the amplitude of systolic cell contraction, reaching 149% of control level, with an apparent ED(50) value of 4.5 nM. The increase in the contraction amplitude was markedly inhibited by pretreatment with verapamil or thapsigargin. An increase in the amplitude of [Ca2+](i) transients, similar to that in the amplitude of cell contraction, was observed in indo-1-loaded cardiomyocytes exposed to 0.1 [mu]M (+)-3-PPP. Exposure to 10 nM of haloperidol or (+)-pentazocine induced an increase in the amplitude of contraction, reaching 188% and 138% (respectively) of control level. A lower concentration of haloperidol or (+)- pentazocine (1 nM) did not induce an increase in the contraction amplitude but rather reduced the amplitude to 70- 80% of control.

108. Bayewitch M., Avidor-Reiss T., Levy R., Barg J., Mechoulam R. and Vogel Z. (1995) The peripheral cannabinoid receptor - adenylate-cyclase inhibition and G-protein coupling. FEBS Lett. 375:143-147.
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Abstract: Two cannabinoid receptors, designated neuronal (or CB1) and peripheral (or CB2), have recently been cloned, Activation of CB1 receptors leads to inhibition of adenylate cyclase and N- type voltage-dependent Ca2+ channels, Here we show, using a CB2 transfected Chinese hamster ovary cell line, that this receptor binds a variety of tricyclic cannabinoid ligands as well. as the endogenous ligand anandamide, Activation of the CB2 receptor by various tricyclic cannabinoids inhibits adenylate cyclase activity and this inhibition is pertussis toxin sensitive indicating that this receptor is coupled to the G(i)/G(o) GTP-binding proteins, Interestingly, contrary to results with CB1, anandamide did not inhibit the CB2 coupled adenylate cyclase activity and Delta(9)-tetrahydrocannabinol had only marginal effects, These results characterize the CB2 receptor as a functional and distinctive member of the cannabinoid receptor family.

109. Barg J., Zimlichman R., Belcheva M., Levy R., Coscia C.J. and Vogel Z. (1995) [kappa]-Opioid agonists inhibit 3H-thymidine incorporation and phosphoinositol (PI) turnover in guinea pig fetal brain cell aggregates. Analgesia 1:268-271.

110. Bayewitch M., Levy R., Avidor-Reiss T., Mechoulam R., Barg J. and Vogel Z. (1995) Adenylate cyclase inhibition by the peripheral cannabinoid receptor. Analgesia 1:286-289.

111. Zimlichman R., Gefel D., Eliahou H., Matas Z., Rosen B., Gass S., Ela C., Eilam Y., Vogel Z. and Barg J. (1995) Development of opioid receptors in hearts of normotensive and hypertensive rats. Analgesia 1:882-885.

112. Heldman E., Barg J., Vogel Z., Pollard H.B. and Zimlichman R. (1996) Correlation between secretagogue-induced Ca2+ influx, intracellular Ca2+ levels and secretion of catecholamines in cultured adrenal chromaffin cells. Neurochem. Int. 28:325-334.
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Abstract: Catecholamine secretion induced by various secretagogues in cultured bovine chromaffin cells has been correlated with Ca2+ influx and intracellular Ca2+ concentrations. Nicotine and high K+ caused prompt secretion of catecholamines from cells. Coincidently, both secretagogues evoked (45)[Ca2+] influx with a parallel increase in free intracellular Ca2+ concentration, as determined by Quin 2 fluorescence. However, the rate of return of Ca2+ level to baseline after nicotine stimulation was more rapid than after K+ stimulation. In comparison, stimulation with veratridine produced a slow and prolonged Ca2+ influx accompanied by lower levels of intracellular Ca2+ than those observed after nicotine or K+ stimulation. Yet, during 15 min of stimulation, veratridine induced a substantial catecholamine release, which was larger than that obtained after nicotine or K+ stimulations. The Ca2+ ionophore A23187 (1 [mu]M) induced a pronounced increase in intracellular Ca2+ levels, but did not evoke any significant catecholamine release. Finally, addition of the Ca2+ channel blocker verapamil following stimulation, at a time when intracellular Ca2+ concentration was at its peak level, did not affect the rate of decline in intracellular free Ca2+ concentration but promptly blocked Ca2+ uptake and catecholamine secretion. These findings suggest that the rate of Ca2+ influx, rather than the absolute level of intracellular Ca2+ concentration, determines the rate and extent of catecholamine release.

113. Heldman E., Barg J., Fisher A., Levy R., Pittel Z., Zimlichman R., Kushnir M. and Vogel Z. (1996) Pharmacological basis for functional selectivity of partial muscarinic receptor agonists. Eur. J. Pharmacol. 297:283-291.
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Abstract: Muscarinic receptor agonists activate phosphoinositide hydrolysis and adenylate cyclase in Chinese hamster ovary cells transfected with cDNAs encoding the human muscarinic m1 and m(3) receptors. Whereas carbachol activates similarly both receptor subtypes, 4-[3-chlorophenyl-carbamoyloxy]-2- butynyltrimethyl ammonium chloride (McN-A-343) preferentially activates the m1 subtype over m3, in regard to both phosphoinositide hydrolysis and adenylate cyclase activity. On the other hand, oxotremorine activates phosphoinositide hydrolysis to a similar extent in both cell lines, but it activates preferentially adenylate cyclase in m1 versus m3 receptor expressing cells. Relative to carbachol, both McN-A- 343 and oxotremorine activate preferentially phosphoinositide hydrolysis over adenylate cyclase in both cell lines. Prolonged incubation of cells with either carbachol, McN-A-343, or oxotremorine down-regulated the m1 receptors. This was accompanied by a parallel decrease in adenylate cyclase activity, whereas phosphoinositide hydrolysis remained relatively high. Inactivation of the receptors by alkylation with acetylethylcholine mustard, or by blocking with atropine, reduced carbachol-stimulated adenylate cyclase activity more effectively than carbachol-induced phosphoinositide hydrolysis in both m1 and m3 receptor expressing cells. These findings imply that the receptor reserve in these cell lines is greater for phosphoinositide hydrolysis response than for adenylate cyclase response. Yet, the receptor reserve for each of these responses is similar in both m1 and m3 receptor expressing cells, Since the binding affinities of McN-A-343 and of oxotremorine to m1 and m3 receptors are very similar, and since both cell lines contain similar amounts of spare receptors, we propose that the preferential activation of muscarinic m1 over m3 receptor by partial agonists is related to differences in the abilities of the two receptor subtypes to undergo conformational changes following agonist binding. This hypothesis is supported by results showing that the muscarinic m1 but not m3 receptor exhibits two affinity states in a competition binding assay.

114. Zimlichman R., Gefel D., Eliahou H., Matas Z., Rosen B., Gass S., Ela C., Eilam Y., Vogel Z. and Barg J. (1996) Expression of opioid receptors during heart ontogeny in normotensive and hypertensive rats. Circulation 93:1020-1025.

Abstract: Background: The opioidergic systems are involved in modulating nociceptive stimuli. In addition, recent results suggest that endogenous and exogenous opioids could play a role in the modulation of blood pressure and cardiac functions. However, little is known regarding the expression and role of opioid- binding sites in the heart. The decreased sensitivity to noxious stimuli in hypertensive rats raises the possibility of different developmental pattern expression of opioid-binding sites in normotensive versus hypertensive rats. Methods and Results: Opioid receptor expression in hearts from hypertensive and normotensive rats was studied during heart development by binding assays. From P1 until P90, the development of the heart in the two rat strains was accompanied by a gradual increase in the density of kappa-opioid receptors. Hearts from hypertensive rats expressed significantly higher levels of kappa receptors compared with those of normotensive rats. At ages older than P7, mu-opioid receptors could not be detected in hearts of both strains, whereas delta-opioid-binding sites gradually increased until reaching adult levels. Seven-day-old cardiomyocyte cultures of both rat strains expressed similar densities of delta or kappa receptors to those observed in hearts from 7- day-old neonates. The mu-binding sites were not detected in cardiomyocyte cultures. Similar to the in vivo state, cultured myocytes from hypertensive rats had significantly higher levels of kappa-binding sites (1.5-fold) compared with those of normotensive rats. The kappa sites are pertussis toxin sensitive, and the state of coupling of the receptor to G protein is similar for the two rat strains. Conclusions: The role of opioid-binding sites in the heart is not completely clear. Hypertensive rats are known to be less sensitive to noxious stimuli compared with normotensive rats. It is controversial whether the site of application of noxious stimuli plays an important role in the sensitivity to pain in hypertensive rats. We suggest that the opioidergic system could play a role in the modulation of blood pressure in addition to its known effect on nociception.

115. Altstein M., Gazit Y., Ben-Aziz O., Gabay T., Marcus R., Vogel Z. and Barg J. (1996) Induction of cuticular melanization in Spodoptera littoralis larvae by PBAN/MRCH: Development of a quantitative bioassay and structure function analysis. Arch. Insect Biochem. Physiol. 31:355-370.

Abstract: PBAN (also termed melanization and reddish coloration hormone, MRCH) is a cerebral factor known to regulate sex pheromone biosynthesis and cuticular melanization in moths. In the present study we developed a quantitative method (based on computerized image analysis of cuticles) to determine the effect of Helicoverpa tea PBAN (Hez-PBAN) on cuticular melanization and to study the structure-activity relationship of the neuropeptide in Spodoptera littoralis larvae. The results indicate that Hez-PBAN stimulates cuticular melanization in an interspecific manner, and that the minimal dose evoking formation of melanins is between 3-10 pmol/larva. Higher doses of Hez-PBAN did not stimulate melanization any further. Examination of the structure-activity relationship of Hez-PBAN revealed that the first eight N-terminal amino acids are not essential for the melanotropic activity and that the activity resides in the C-terminal region. Within this region the C-terminal amide was found to play a very important role. (C) 1996 Wiley-Liss, Inc.

116. Belcheva M.M., Ho M.T., Ignatova E.G., Jefcoat L.B., Barg J., Vogel Z., McHale R.J., Johnson F.E. and Coscia C.J. (1996) Buprenorphine differentially alters opioid receptor adaptation in rat brain regions. J. Pharmacol. Exp. Ther. 277:1322-1327.

Abstract: Previous in vivo studies revealed that the mixed agonist- antagonist buprenorphine can down-regulate mu and up-regulate delta(2) and kappa(1), opioid receptors in rat brain, In this report brain regional differences in opioid receptor adaptation were addressed. Rats received i.p. injections with buprenorphine (0.5-2.5 mg/kg) and were killed 20 h later. Membranes from 7 brain regions-were analyzed for mu (H-3-[D- Ala(2),N-mephe(4),Gly-ol(5)] enkephalin), kappa(1) (H-3-U- 69593), delta(1) (H-3-[D-Pen(2),D-Pen(5)] enkephalin) and delta(2) (H-3-deltorphin II) receptor binding parameters. Buprenorphine induced down-regulation of mu receptors in frontal cortex, occipital cortex, thalamus, hippocampus, striatum and brain stem. K-d values for H-3-[D-Ala(2),N- mephe(4),Gly-ol(5)] enkephalin were unchanged from controls. Up-regulation of kappa(1) receptors was observed in frontal, parietal, occipital cortexes and striatum. Binding to delta(2) sites was elevated in frontal and parietal cortexes. Buprenorphine did not alter delta(1) binding in any of the regions examined. Changes in opioid receptor adaptation induced by buprenorphine were further supported by data from crosslinking of I-125-delta-endorphin io cortical membrane preparations, A reduction in a 60- to 65-kDa band was detected in frontal and occipital cortices in which binding assays revealed down-regulation of mu receptors. In parietal cortex neither the 60- to 65-kDa product nor B-max changes were observed. These results indicate that buprenorphine is a useful tool to study brain opioid receptor adaptation in vivo and the information accrued may be relevant to the mode of action of this drug in the treatment of heroin and cocaine abuse.

117. Bayewitch M., Rhee M.-H., Avidor-Reiss T., Breuer A., Mechoulam R. and Vogel Z. (1996) (-)-[Delta]9-Tetrahydrocannabinol antagonizes the peripheral cannabinoid receptor-mediated inhibition of adenylyl cyclase. J. Biol. Chem. 271:9902-9905.
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Abstract: (-)-Delta(9)-Tetrahydrocannabinol ((-)-Delta(9)-THC) is the major active psychotropic component of the marijuana plant, Cannabis sativa. The membrane proteins that have been found to bind this material or its derivatives have been called the cannabinoid receptors. Two GTP-binding protein-coupled cannabinoid receptors have been cloned. CB1 or the neuronal cannabinoid receptor is found mostly in neuronal cells and tissues while CB2 or the peripheral cannabinoid receptor has been detected in spleen and in several cells of the immune system. It has previously been shown that activation of CB1 or CB2 receptors by cannabinoid agonists inhibits adenylyl cyclase activity. Utilizing Chinese hamster ovary cells and COS cells transfected with the cannabinoid receptors we report that (-)- Delta(9)-THC binds to both receptors with similar affinity. However, in contrast to its capacity to serve as an agonist for the CB1 receptor, (-)-Delta(9)-THC was only able to induce a very slight inhibition of adenylyl cyclase at the CB2 receptor. Morever, (-)-Delta(9)-THC antagonizes the agonist-induced inhibition of adenylyl cyclase mediated by CB2. Therefore, we conclude that (-)-Delta(9)-THC constitutes a weak antagonist for the CB2 receptor.

118. Matus-Leibovitch N., Vogel Z., Ezra-Macabee V., Etkin S., Nevo I. and Attali B. (1996) Chronic morphine administration enhances the expression of Kv1.5 and Kv1.6 voltage-gated K+ channels in rat spinal cord. Mol. Brain Res. 40:261-270.
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Abstract: Prolonged opiate administration leads re, the development of tolerance and dependence. These phenomena are accompanied by selective regulation of distinct cellular proteins and mRNAs, including ionic channels. Acute opiate administration differentially affects voltage-dependent K+ currents. Whereas, opiate activation of K+ channels is well established opioid- induced inhibition of K+ conductance has also been studied. In this study, we focused on the effect of chronic morphine exposure on voltage-dependent Shaker-related Kv1.5 and Kv1.6 K+ channel gene expression and on Kv1.5 protein levels in the rat spinal cord. Several experimental approaches including in-situ hybridization, RNAse protection, reverse transcriptase- polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry were employed. We found that motor neurons are highly enriched in Kv1.5 and Kv1.6 mRNA and in Kv1.5 channel protein. Moreover, we found significant increases in the amount of mRNA encoding for these two K+ channels and in Kv1.5 channel protein in the spinal cord of morphine-treated rats, compared with controls. For example, quantitative in-situ hybridization, revealed a 2.1 +/- 0.15- and 2.3 +/- 0.5-fold increase in Kv1.5 and Kv1.6 channel mRNA levels, respectively. Similar results were obtained by semiquantitative RT-PCR analyses. Kv1.5 protein level was increased by 1.9-fold in the spinal cord of morphine-treated rats. Our results suggest that Kv1.5 and Kv1.6 Shaker K+ channels play an important role in regulating motor activity and that increases in mRNA and protein levels of the spinal cord K+ channels after chronic morphine exposure could be viewed as a cellular adaptation which compensates for a persistent opioid-induced inhibition of K+ channel activity. These alterations may account, in part, for the cellular events leading to opiate tolerance and dependence.

119. Avidor-Reiss T., Nevo I., Levy R., Pfeuffer T. and Vogel Z. (1996) Chronic opioid treatment induces adenylyl cyclase V superactivation - Involvement of G[beta][gamma]. J. Biol. Chem. 271:21309-21315.
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Abstract: It has been known for some time that chronic treatment of neuronal cells and tissues with opioids, contrary to their acute effect, leads to an increase in cAMP accumulation. This phenomenon, defined as adenylyl cyclase superactivation, has been implicated in opiate addiction, yet the mechanism by which it is induced remains unclear. Here, we show that this phenomenon can be reproduced and studied in COS-7 cells cotransfected with adenylyl cyclase type V and mu-opioid receptor cDNAs. These cells display acute opioid inhibition of adenylyl cyclase activity, whereas prolonged exposure to the mu-agonist morphine or [D-Ala(2), N-methyl-Phe(4), Gly- ol(5)]enkephalin leads to a time-dependent superactivation of adenylyl cyclase. This superactivated state is reversible, because it is gradually lost following agonist withdrawal, Adenylyl cyclase superactivation can be prevented by pertussis toxin pretreatment, indicating the involvement of G(i/o) proteins, or by cotransfection with the carboxyl terminus of beta-adrenergic receptor kinase or with alpha-transducin (scavengers of G(beta gamma) dimers), indicating a role for the G protein beta gamma dimers in adenylyl cyclase superactivation. However, contrary to several other G(beta gamma)-dependent signal transduction mechanisms (e.g. the extracellular signal-regulated kinase 2/MAP kinase pathway), adenylyl cyclase superactivation is not affected by the Has dominant negative mutant N17-Ras.

120. Fisher A., Heldman E., Gurwitz D., Haring R., Karton Y., Meshulam H., Pittel Z., Marciano D., Brandeis R., Sadot E., Barg Y., Pinkas-Kramarski R., Vogel Z., Ginzburg I., Treves T.A., Verchovsky R., Klimowsky S. and Korczyn A.D. (1996) M1 agonists for the treatment of Alzheimer's disease - Novel properties and clinical update, in Neurobiology of Alzheimer's Disease 777, pp. 189-196.

Abstract: The AF series compounds, AF102B and congeners of AP150(S), are functionally selective agonists for ml muscarinic receptors (mlAChRs). This is shown in stable transfected CHO and PC12 cells (PC12M1) with mlm5AChRs and mlAChRs, respectively. AF102B and AF150(S) are partial agonists, but AF150, AF151, and AF151(S) are full agonists in stimulating phosphoinositides hydrolysis or arachidonic acid release in these cells. Yet, all these compounds behave as antagonists when compared with carbachol in elevating cAMP levels. In PC12M1 cells, unlike carbachol, the AP series compounds induce only minimal to moderate neurite outgrowth. Yet, these agonists synergize strongly with NGF, which by itself mediates only a mild response. Stimulation of mlAChRs by AF102B, AF150(S) and AF151(S) in PC12M1 cells enhances secretion of beta/A4 amyloid precursor protein derivatives (APPs). The enhanced APPs secretion induced by AF102B is potentiated by NGF. AF102B also stimulates APPs secretion from rat cortical slices. Stimulation of mlAChR in PC12M1 cells with carbachol or AP102B decreases tau phosphorylation as indicated by specific tau-1 mAb and alkaline phosphatase treatment. Due to the above mentioned properties ml agonists may be of unique value in delaying the progression of Alzheimer's disease (AD). The AF series compounds show a wide safety margin and improve memory and learning deficits in animal models for AD. There is a dearth of clinical reports on ml agonists. These include studies on AF102B and xanomeline, another ml selective agonist. We tested AF102B in escalating doses of 20, 40, 60 mg, tid, po, (each dose for 2 weeks) for a total of 10 weeks. This was a single- blind placebo-controlled, parallel-group study in patients with probable AD. AF102B was significantly effective at 40 and 60 mg, tid in the ADAS, ADAS-cognitive and ADAS-word recognition scales.

121. Mechoulam R., Ben-Shabat S., Hanus L., Fride E., Bayewitch M. and Vogel Z. (1996) Endogenous cannabinoid ligands. Adv. Exp. Med. Biol. 402:95-101.

122. Mechoulam R., Ben-Shabat S., Hanus L., Fride E., Vogel Z., Bayewitch M. and Sulcova A.E. (1996) Endogenous cannabinoid ligands - Chemical and biological studies. J. Lipid Med. Cell Signal. 14:45-49.

Abstract: Arachidonic acid ethanolamide (anandamide) is a brain constituent that binds to the brain cannabinoid receptor (CB1). It produces many of the pharmacological effects caused by Delta(9)-tetrahydrocannabinol (Delta(9)-THC) in mice. Anandamide parallels Delta(9)-THC in its specific interaction with the cannabinoid receptor and in inhibition of adenylate cyclase. Two additional fatty acid ethanolamides that bind to the cannabinoid receptor, homo-gamma-linolenylethanolamide and docostetraenylethanolamide, have been identified in the brain. We believe that the anandamides are involved in the coordination of movement and short term memory. Depression of ambulation in an open field and the analgetic response to anandamide are not fully developed until adulthood, possibly due to an age-related increase in the CB1 receptor concentration. This observation has clinical implications in pediatrics. A second cannabinoid receptor (CB2) is present in the spleen. A monoglyceride, 2-arachidonyl-glycerol which binds to both CB1 and CB2 in transfected cells and inhibits andenylate cyclase in spleen cells was found in the gut. Its role is apparently associated with the immune system. These fatty acids amides and esters represent a new family of chemical modulators in the body.

123. Shohami E., Weidenfeld J., Ovadia H., Vogel Z., Hanus L., Fride E., Breuer A., Ben-Shabat S., Sheskin T. and Mechoulam R. (1996) Endogenous and synthetic cannabinoids: recent advances. CNS Drug Rev. 2:429-451.

124. Matus-Leibovitch N., Nevo I. and Vogel Z. (1997) Differential distribution of synapsin IIa and IIb mRNAs in various brain structures and the effect of chronic morphine administration on the regional expression of these isoforms. Mol. Brain Res. 45:301-316.
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Abstract: Quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization techniques were used to determine the regional distribution of synapsin IIa and IIb mRNAs in rat central nervous system and to assess the effect of chronic morphine administration on the gene expression of these two isoforms of synapsin II. These isoforms are members of a family of neuron-specific phosphoproteins thought to be involved in the regulation of neurotransmitter release. Our data demonstrate the widespread distribution, yet regionally variable expression, of synapsin IIa and IIb mRNAs throughout the adult rat brain and spinal cord. The ratios of the relative abundance of synapsins IIa and IIb differed by up to 4.5-fold among the various regions studied. Synapsin IIa and IIb mRNAs were shown to be highly concentrated in the thalamus and in the hippocampus, whereas lower concentrations were found in most other central nervous system structures. In this study, we show differential regulation by morphine of synapsins IIa and IIb in various regions of the brain. In the striatum, a 2.4-fold increase was observed in the levels of synapsin IIa mRNA following chronic morphine regime, whereas no change was found for synapsin IIb. On the other hand, mRNA levels of synapsin IIb in spinal cord of chronically treated rats were markedly decreased (by 62%), while no alterations were observed in synapsin IIa. Selective regulation by morphine has also been demonstrated in several other central nervous system structures. The opiate-induced regulation of the gene expression of synapsin II isoforms could be viewed as one of the cellular adaptations to the persistent opiate effects and may be involved in the molecular mechanism underlying opiate tolerance and/or dependence.

125. Avidor-Reiss T., Nevo I., Saya D., Bayewitch M. and Vogel Z. (1997) Opiate-induced adenylyl cyclase superactivation is isozyme-specific. J. Biol. Chem. 272:5040-5047.
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Abstract: While acute activation of inhibitory G(i/o)-coupled receptors leads to inhibition of adenylyl cyclase, chronic activation of such receptors leads to an increase in cAMP accumulation. This phenomenon, observed in many cell types, has been referred to as adenylyl cyclase superactivation, At this stage, the mechanism leading to adenylyl cyclase superactivation and the nature of the isozyme(s) responsible for this phenomenon are largely unknown. Here we show that transfection of adenylyl cyclase isozymes into COS-7 cells results in an isozyme- specific increase in AC activity upon stimulation (e.g. with forskolin, ionomycin, or stimulatory receptor ligands). However, independently of the method used to activate specific adenylyl cyclase isozymes, acute activation of the mu-opioid receptor inhibited the activity of adenylyl cyclases I, V, VI, and VIII, while types II, IV, and VII were stimulated and type III was not affected. Chronic mu-opioid receptor activation followed by removal of the agonist was previously shown, in transfected COS-7 cells, to induce superactivation of adenylyl cyclase type V. Here we show that it also leads to superactivation of adenylyl cyclase types I, VI, and VIII, but not of type II, III, IV, or VII, demonstrating that the superactivation is isozyme-specific. Not only were isozymes II, IV, and VII not superactivated, but a reduction in the activities of these isozymes was actually observed upon chronic opiate exposure. These results suggest that the phenomena of tolerance and withdrawal involve specific adenylyl cyclase isozymes.

126. Sheskin T., Hanus L., Slager J., Vogel Z. and Mechoulam R. (1997) Structural requirements for binding of anandamide-type compounds to the brain cannabinoid receptor. J. Med. Chem. 40:659-667.
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Abstract: In order to establish the structural requirements for binding to the brain cannabinoid receptor (CB1), we have synthesized numerous fatty acid amides, ethanolamides, and some related simple derivatives and have determined their K-i values. A few alpha-methyl- or alpha,alpha-dimethylarachidonoylalkylamides were also examined. In the 20:4, n-6 series, the unsubstituted amide is inactive; N-monoalkylation, at least up to a branched pentyl group, leads to significant binding. N,N-Dialkylation, with or without hydroxylation on one of the alkyl groups, leads to elimination of activity. Hydroxylation of the N-monoalkyl group at the omega carbon atom retains activity. In the 20:x, n-6 series, x has to be either 3 or 4; the presence of only two double bonds leads to inactivation. In the n-3 series, the limited data reported suggest that the derived ethanolamides are either inactive or less active than comparable compounds in the n-6 series. Alkylation or dialkylation of the alpha carbon adjacent to the carbonyl group retains the level of binding in the case of anandamide (compounds 48, 49); however, alpha- monomethylation or alpha,alpha-dimethylation of N-propyl derivatives (50-53) potentiates binding and leads to the most active compounds seen in the present work (K-i values of 6.9 +/- 0.7 to 8.4 +/- 1.1 nM). We have confirmed that the presence of a chiral center on the N-alkyl substituent may lead to enantiomers which differ in their levels of binding (compounds 54, 57 and 55, 56).

127. Ela C., Barg J., Vogel Z., Hasin Y. and Eilam Y. (1997) Distinct components of morphine effects on cardiac myocytes are mediated by the [kappa] and [delta] opioid receptors. J. Mol. Cell. Cardiol. 29:711-720.
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Abstract: Morphine exerts direct effects on cultured cardiac myocytes from neonatal rats. These effects are mediated via the delta and the kappa opioid receptors, as mu opioid receptors are not present in neonatal cardiomyocyte cultures. Binding parameters to the delta and kappa opioid receptors were determined in membrane preparations from these cultures by heterologous competition to [H-3]diprenorphine binding, with [D-Pen(2), D- Pen(5)]-enkephalin (DPDPE) and trans-(dl)-3,4-dichloro-N- methyl-N- [2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate (U-50,458H) as specific displacers respectively. To define the components of morphine effects mediated via activation. of either the delta or the kappa opioid receptor alone, cardiac myocytes were exposed to morphine in the presence of specific antagonists to the kappa or delta opioid receptor respectively. Activation of the kappa opioid receptors by morphine caused a transient increase in Ca2+ influx, leading to increase in amplitudes of [Ca2+](i) transients and contraction, with no change in the intracellular pH. Activation of the delta opioid receptors alone by morphine caused a decrease in the amplitude of contraction. This decrease was mediated by a decrease in the intracellular pH leading to reduced responsiveness of the myofilaments to Ca2+. There was no change in Ca2+ influx and in the amplitude of [Ca2+](i) transients. The effects mediated through the delta opioid but not through the kappa opioid receptors were pertussis toxin sensitive, indicating coupling of the delta opioid receptors to pertussis toxin sensitive GTP-binding proteins. The overall effects of morphine on the neonatal cardiac myocytes were the sum of the effects exerted by morphine when it activated each of the opioid receptors alone. (C) 1997 Academic Press Limited.

128. Mechoulam R., Fride E., Hanus L., Sheskin T., Bisogno T., DiMarzo V., Bayewitch M. and Vogel Z. (1997) Anandamide may mediate sleep induction. Nature 389:25-26.
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Abstract:In rodents, the Ras-specific guanine-nucleotide exchange factor (Ras-GRF) is expressed in different areas of the brain and, at a reduced level, also in the spinal cord. No expression of the 140 kDa Ras-GRF was detected in dorsal root ganglia and all other tissues tested. Analysis of primary cultures derived from brain reveals that this exchange factor is only present in neurons of the central nervous system. In primary hippocampal cultures, the expression of Ras-GRF increases in parallel with the onset of a neuronal network and in the whole brain it increases sharply after birth.

129. Rhee M.-H., Vogel Z., Barg J., Bayewitch M., Levy R., Hanus L., Breuer A. and Mechoulam R. (1997) Cannabinol derivatives: Binding to cannabinoid receptors and inhibition of adenylylcyclase. J. Med. Chem. 40:3228-3233.
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Abstract: Several derivatives of cannabinol anti the 1,1-dimethylheptyl homolog (DMM) of cannabinol were prepared and assayed for binding to the brain and the peripheral cannabinoid receptors (CB1 and CB2), as well as for activation of CB1- and CB2- mediated inhibition of adenylylcyclase. The DMH derivatives were much more potent than the pentyl (i.e., cannabinol) derivatives. 11-Hydroxycannabinol (4a) was found to bind potently to both CB1 and CB2 (K-i values of 38.0 +/- 7.2 and 26.6 +/- 5.5 nM, respectively) and to inhibit CB1-mediated adenylylcyclase with an EC50 of 58.1 +/- 6.2 nM but to cause only 20% inhibition of CB2-mediated adenylylcyclase at 10 [mu]M. It behaves as a specific, though not potent, CB2 antagonist. 11-Hydroxycannabinol-DMH (4b) is a very potent agonist for both CB1 and CB2 (K-i values of 100 +/- 50 and 200 +/- 40 pM; EC50 of adenylylcyclase inhibition 56.2 +/- 4.2 and 207.5 +/- 27.8 pM, respectively).

130. Zippel R., Gnesutta N., Matus-Leibovitch N., Mancinelli E., Saya D., Vogel Z. and Sturani E. (1997) Ras-CRF, the activator of Ras, is expressed preferentially in mature neurons of the central nervous system. Mol. Brain Res. 48:140-144.
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Abstract:In rodents, the Ras-specific guanine-nucleotide exchange factor (Ras-GRF) is expressed in different areas of the brain and, at a reduced level, also in the spinal cord. No expression of the 140 kDa Ras-GRF was detected in dorsal root ganglia and all other tissues tested. Analysis of primary cultures derived from brain reveals that this exchange factor is only present in neurons of the central nervous system. In primary hippocampal cultures, the expression of Ras-GRF increases in parallel with the onset of a neuronal network and in the whole brain it increases sharply after birth.

131. Pintov S., Lahat E., Alstein M., Vogel Z. and Barg J. (1997) Acupuncture and the opioid system: Implications in management of migraine. Pediatric Neurol. 17:129-133.

Abstract: We investigated the effectiveness of acupuncture in childhood migraine in 22 children with migraine, randomly divided into two groups: a true acupuncture group (12 children) and a placebo acupuncture group (10 children), Ten healthy children served as a control group, Opioid activity in blood plasma was assayed by two methods: (1) determination of total (panopioid) activity with an opiate radioreceptor assay, and (2) determination of beta-endorphinlike immunoreactivity by radioimmunoassay, The true acupuncture treatment led to significant clinical reduction in both migraine frequency and intensity, At the beginning of the study, significantly greater panopioid activity was evident in plasma of the control group than in plasma of the migraine group, The true acupuncture group showed a gradual increase in the panopioid activity in plasma, which correlated with the clinical improvement, After the tenth treatment, the values of opioid activity of the true acupuncture group were similar to those of the control group, whereas the plasma of the placebo acupuncture group exhibited insignificant changes in plasma panopioid activity, In addition, a significant increase in beta-endorphin levels was observed in the migraine patients who were treated in the true acupuncture group as compared with the values before treatment or with the values of the placebo acupuncture group, The results suggest that acupuncture may be an effective treatment in children with migraine headaches and that it leads to an increase in activity of the opioidergic system. (C) 1997 by Elsevier Science Inc. All rights reserved.

132. Belcheva M.M., Vogel Z., Ignatova E., Avidor-Reiss T., Zippel R., Levy R., Young E.C., Barg J. and Coscia C.J. (1998) Opioid modulation of extracellular signal-regulated protein kinase activity is Ras-dependent and involves G[beta][gamma] subunits. J. Neurochem. 70:635-645.

Abstract: Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with mu-, delta-, or kappa-opioid receptors and ERK1- or ERK2-containing plasmids, Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding mu- ([D- Ala(2),Me-Phe(4),Gly-ol(5)]enkephalin)-, delta- ([D-Pen(2),D- Pen(5)] enkephalin)-, or kappa- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed, This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a beta gamma scavenger, CD8-beta-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2, When epidermal growth factor was used to activate ERK1,chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor, This inhibition was blocked by the corresponding antagonists and CD8-beta-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and beta gamma subunits of Gi/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.

133. Bayewitch M.L., Avidor-Reiss T., Levy R., Pfeuffer T., Nevo I., Simonds W.F. and Vogel Z. (1998) Differential modulation of adenylyl cyclases I and II by various G[beta] subunits. J. Biol. Chem. 273:2273-2276.
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Abstract: The accepted dogma concerning the regulation of adenylyl cyclase (AC) activity by G(beta gamma) dimers states that the various isoforms of AC respond differently to the presence of free G(beta gamma) It has been demonstrated that AC I activity is inhibited and AC II activity is stimulated by G(beta gamma) subunits, This result does not address the possible G differences in modulation that may exist among the different G(beta gamma) heterodimers, Six isoforms of G(beta) and 12 isoforms of G(gamma) have been cloned to date, We have established a cell transfection system in which G(beta) and G(gamma) cDNAs were cotransfected with either AC isoform I or II and the activity of these isoforms was determined, We found that while AC I activity was inhibited by both G(beta 1/gamma 2) and G(beta 5/gamma 2) combinations, AC II responded differentially and was stimulated by G(beta 1/gamma 2). This finding demonstrates differential modulatory activity by different combinations of G(beta gamma) on the same AC isoform and demonstrates another level of complexity within the AC signaling system.

134. Zangen A., Herzberg U., Vogel Z. and Yadid G. (1998) Nociceptive stimulus induces release of endogenous [beta]-endorphin in the rat brain. Neuroscience 85:659-662.
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Abstract: The hypothesis that the naturally occurring analgesic peptide, beta-endorphin, is released in the brain in response to pain had never been directly validated. In this study, we applied a brain microdialysis method for monitoring beta-endorphin release in vivo, to test this hypothesis in the brains of conscious, freely moving rats. Herein we first show that endogenous beta-endorphin can be measured in vivo in the brain under physiological conditions. Upon induction of a nociceptive stimulus by injection of formalin into the hind-paws of rats, the extracellular levels of beta-endorphin in their arcuate nucleus increased by 88%, corresponding to their nociceptive response, This direct evidence for the release of endogenous beta-endorphin in the brain in response to nociceptive stimulus indicates a possible mechanism for organisms to cope with pain. (C) 1998 IBRO. Published by Elsevier Science Ltd.

135. Ben-Shabat S., Fride E., Sheskin T., Tamiri T., Rhee M.-H., Vogel Z., Bisogno T., De Petrocellis L., Di Marzo V. and Mechoulam R. (1998) An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity. Eur. J. Pharmacol. 353:23-31.
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Abstract: 2-Arachidonoyl-glycerol (2-Ara-G1) has been isolated from various tissues and identified as an endogenous ligand for both cannabinoid receptors, CB1 and CB2. Here we report that in spleen, as in brain and gut, 2-Ara-G1 is accompanied by several 2-acyl-glycerol esters, two major ones being 2-linoleoyl- glycerol (2-Lino-G1) and 2-palmitoyl-glycerol (2-Palm-G1). These two eaters do not bind to the cannabinoid receptors, nor do they inhibit adenylyl cyclase via either CB1 or CB2; however, they significantly potentiate the apparent binding of 2-Ara-G1 and its apparent capacity to inhibit adenylyl cyclase. Together these eaters also significantly potentiate 2-Ara-G1 inhibition of motor behavior, immobility on a ring, analgesia on a hot plate and hypothermia caused by 2-Ara-G1 in mice. 2- Lino-G1, but not 2-Palm-G1, significantly inhibits the inactivation of 2-Ara-G1 by neuronal and basophilic cells. These data indicate that the biological activity of 2-Ara-G1 can be increased by related, endogenous 2-acyl-glycerols, which alone show no significant activity in any of the tests employed, This effect ('entourage effect') may represent a novel route for molecular regulation of endogenous cannabinoid activity. (C) 1998 Elsevier Science B.V. All rights reserved.

136. Bayewitch M. L., Avidor-Reiss T., Levy R., Pfeuffer T., Nevo I., Simonds W. F. and Vogel Z. (1998) Inhibition of adenylyl cyclase isoforms V and VI by various G[beta][gamma] subunits. FASEB J. 12:1019-1025.
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Abstract: An intriguing development in the G-protein signaling field has been the finding that not only the G(alpha) subunit, but also G(beta gamma) subunits, affect a number of downstream target molecules. One of the downstream targets of G(beta gamma) is adenylyl cyclase, and it has been demonstrated that a number of isoforms of adenylyl cyclase can be either inhibited or stimulated by G(beta gamma) subunits. Until now, adenylyl cyclase type I has been the only isoform reported to be inhibited by free G(beta gamma). Here we show by transient cotransfection into COS-7 cells of either adenylyl cyclase V or VI, together with G gamma(gamma 2) and various G(beta) subunits, that these two adenylyl cyclase isozymes are markedly inhibited by G(beta gamma). In addition, we show that G(beta 1) and G(beta 5) subunits differ in their activity. G(beta 1) transfected alone markedly inhibited adenylyl cylcase V and VI (probably by recruiting endogenous G(gamma) subunits). On the other hand, G(beta 5) produced less inhibition of these isozymes, and its activity was enhanced by the addition of G(gamma 2). These results demonstrate that adenylyl cyclase types V and VI are inhibited by G(beta gamma) dimers and that G(beta 1) and G(beta 5) Subunits differ in their capacity to regulate these adenylyl cyclase isozymes.

137. Rhee M.-H., Bayewitch M., Avidor-Reiss T., Levy R. and Vogel Z. (1998) Cannabinoid receptor activation differentially regulates the various adenylyl cyclase isozymes. J. Neurochem. 71:1525-1534.

Abstract: Two cannabinoid receptors belonging to the superfamily of G protein-coupled membrane receptors have been identified and cloned: the neuronal cannabinoid receptor (CB1) and the peripheral cannabinoid receptor (CB2). They have been shown to couple directly to the G(i/o) subclass of G proteins and to mediate inhibition of adenylyl cyclase upon binding of a cannabinoid agonist. In several cases, however, cannabinoids have been reported to stimulate adenylyl cyclase activity, although the mechanism by which they did so was unclear. With the cloning of nine adenylyl cyclase isozymes with various properties, including different sensitivities to alpha(s), alpha(i/o), and py subunits, it became important to assess the signaling pattern mediated by each cannabinoid receptor via the different adenylyl cyclase isozymes. In this work, we present the results of cotransfection experiments between the two types of cannabinoid receptors and the nine adenylyl cyclase isoforms. We found that independently of the method used to stimulate specific adenylyl cyclase isozymes (e.g., ionomycin, forskolin, constitutively active alpha(s), thyroid-stimulating hormone receptor activation), activation of the cannabinoid receptors CB1 and CB2 inhibited the activity of adenylyl cyclase types I, V, VI, and VIII, whereas types II, IV, and VII were stimulated by cannabinoid receptor activation. The inhibition of adenylyl cyclase type III by cannabinoids was observed only when forskolin was used as stimulant. The activity of adenylyl cyclase type IX was inhibited only marginally by cannabinoids.

138. Nevo I., Avidor-Reiss T., Levy R., Bayewitch M., Heldman E. and Vogel Z. (1998) Regulation of adenylyl cyclase isozymes on acute and chronic activation of inhibitory receptors. Mol. Pharmacol. 54:419-426.
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Abstract: Adenylyl cyclase superactivation, a phenomenon by which chronic activation of inhibitory G(i/o)-coupled receptors leads to an increase in cAMP accumulation, is believed to play an important role as a compensatory response of the cAMP signaling system in the cell. However, to date, the mechanism by which adenylyl cyclase activity is regulated by chronic exposure to inhibitory agonists and the nature of the adenylyl cyclase isozymes participating in this process remain largely unknown. Here we show, using COS-7 cells transfected with the various AC isozymes, that acute activation of the D-2 dopaminergic and m4 muscarinic receptors inhibited the activity of adenylyl cyclase isozymes I, V, VI, and VIII, whereas types II, IV, and VII were stimulated and type III was not affected. Conversely, chronic receptor activation led to superactivation of adenylyl cyclase types I, V, VI, and VIII and to a reduction in the activities of types II, IV, and VII. The activity of AC-III also was reduced. This pattern of inhibition/stimulation of the various adenylyl cyclase isozymes is similar to that we recently observed on acute and chronic activation of the mu-opioid receptor, suggesting that isozyme-specific adenylyl cyclase superactivation may represent a general means of cellular adaptation to the activation of inhibitory receptors and that the presence/absence and intensity of the adenylyl cyclase response in different brain areas (or cell types) could be explained by the expression of different adenylyl cyclase isozyme types in these areas.

139. Spetea M., Otvos F., Toth G., Nguyen T. M. D., Schiller P. W., Vogel Z. and Borsodi A. (1998) Interaction of agonist peptide [H-3]Tyr-D-Ala-Phe-Phe-NH2 with [mu]-opioid receptor in rat brain and CHO-[mu]/1 cell line. Peptides 19:1091-1098.
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Abstract: Opioid receptor binding properties of [H-3]Tyr-D-Ala-Phe-Phe- NH, (TAPP) were characterized in rat brain and Chinese hamster ovary (CHO) cells expressing the rat CL-receptor. In rat brain, [H-3]TAPP labeled a single class of opioid sites with a dissociation constant (K-d) of 0.31 nM and maximal number of binding sites (B-max) of 119 fmol/mg protein. In CHO-mu/l cell membranes, the K-d and B-max values were 0.78 nM and 1806 fmol/mg protein, respectively. Binding to rat brain was demonstrated to be pharmacologically identical to that obtained with CHO-mu/l cell membranes and modulated by Na+ ions and guanine nucleotides. The high affinity and selectivity of [H- 3]TAPP together with its low non-specific binding make this radioligand a useful tool for labeling the native and cloned mu-opioid receptor. (C) 1998 Elsevier Science Inc.

140. Obadiah J., Avidor-Reiss T., Fishburn C.S., Carmon S., Bayewitch M., Vogel Z., Fuchs S. and Levavi-Sivan B. (1999) Adenylyl cyclase interaction with the D2 dopamine receptor family: differential coupling to Gi, Gz and Gs Cell. Mol. Neurobiol. 19:653-664.

Abstract:1. The D2-type dopamine receptors are thought to inhibit adenylyl cyclase (AC), via coupling to pertussis toxin (PTX)-sensitive G proteins of the Gi family. We examined whether and to what extent the various D2 receptors (D2S, D2L, D3S, D3L, and D4) couple to the PTX-insensitive G protein Gz, to produce inhibition of AC activity. 2. COS-7 cells were transiently transfected with the individual murine dopamine receptors alone, as well as together with the alpha subunit of Gz. PTX treatment was employed to inactivate endogenous alpha i, and coupling to Gi and Gz was estimated by measuring the inhibition of cAMP accumulation induced by quinpirole, in forskolin-stimulated cells. 3. D2S or D2L receptors can couple to the same extent to Gi and to Gz. The D4 dopamine receptor couples preferably to Gz, resulting in about 60% quinpirole-induced inhibition of cAMP accumulation. The D3S and D3L receptor isoforms couple slightly to Gz and result in 15 and 30% inhibition of cAMP accumulation, respectively. 4. We have demonstrated for the first time that the two D3 receptor isoforms, and not any of the other D2 receptor subtypes, also couple to Gs in both COS-7 and CHO transfected cells, in the presence of PTX. 5. Thus, the differential coupling of the D2 dopamine receptor subtypes to various G proteins may add another aspect to the diversity of dopamine receptor function.

141. Nevo I., Avidor-Reiss T., Levy R., Bayewitch M. and Vogel Z. (2000) Acute and chronic activation of the [mu]-opioid receptor with the endogenous ligand endomorphin differentially regulates adenylyl cyclase isozymes Neuropharmacol. 39:364-371.
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Abstract: While acute activation of Gi/o-coupled receptors leads to inhibition of adenylyl cyclase, chronic activation of such receptors produces an increase in cyclic AMP accumulation, particularly evident upon withdrawal of the inhibitory agonist. This phenomenon has been referred to as adenylyl cyclase superactivation and is believed to play an important role in opiate addiction. Nine adenylyl cyclase isozymes have been recently identified and shown by us to be differentially regulated by acute and chronic inhibitory receptor activation. Using COS-7 cells cotransfected with various adenylyl cyclase isozymes, we examined here whether the endomorphins (the most recently discovered of the four classes of endogenous opioid peptides, and which interact selectively with the [mu] receptor) are able to induce inhibition/superactivation of representatives from the various adenylyl cyclase isozyme classes. Here, we show that adenylyl cyclase types I and V were inhibited by acute endomorphin application and superactivated upon chronic exposure, while adenylyl cyclase type II was stimulated by acute and "superinhibited" by chronic endomorphin exposure. These results show that the endomorphins are capable of regulating adenylyl cyclase activity and that different adenylyl cyclase isozymes respond differently to these endogenous ligands.

142. Rhee M.-H., Nevo I., Levy R. and Vogel Z. (2000) Role of the highly conserved Asp-Arg-Tyr motif in signal transduction of the CB2 cannabinoid receptor. FEBS Lett. 466:300-304.
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Abstract: The DRY motif, at the junction of transmembrane helix 3 and intracellular loop 2 of G protein-coupled receptors, is highly conserved. Mutations were introduced into the CB2 cannabinoid receptor to study the role of this motif in CB2 signaling. D mutations (DRY130-132AAA and D130A) markedly reduced binding of cannabinoid agonists, while no significant reduction was observed with R131A or Y132A. Mutating R (R131A) only partially reduced, and mutating Y (Y132A) more efficiently reduced the cannabinoid-induced inhibition of adenylyl cyclase. Thus, in CB2, D130 is involved in agonist binding, whereas Y seems to have a role in receptor downstream signaling.

143. Bayewitch M.L., Nevo I., Avidor-Reiss T., Levy R., Simonds W.F. and Vogel Z. (2000) Alterations in detergent solubility of heterotrimeric G proteins after chronic activation of Gi/o-coupled receptors: Changes in detergent solubility are in correlation with the onset of adenylyl cyclase superactivation. Mol. Pharmacol. 57:820-825.
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Abstract: Prolonged Gi/o protein-coupled receptor activation has been shown to lead to receptor internalization and receptor desensitization. In addition, it is well established that although acute activation of these receptors leads to inhibition of adenylyl cyclase (AC), long-term activation results in increased AC activity (especially evident on removal of the inhibitory agonist), a phenomenon defined as AC superactivation or sensitization. Herein, we show that chronic exposure to agonists of Gi-coupled receptors also leads to a decrease in cholate detergent solubility of G protein subunits, and that antagonist treatment after such chronic agonist exposure leads to a time-dependent reversal of the cholate insolubility. With Chinese hamster ovary and COS cells transfected with several Gi/o-coupled receptors (i.e., mu- and kappa-opioid, and m4-muscarinic), we observed that although no overall change occurred in total content of Galpha-i- and beta1-subunits, chronic agonist treatment led to a marked reduction in the ability of 1% cholate to solubilize Gbeta-gamma as well as Galpha-i. This solubility shift is exclusively observed with Galpha-i, and was not seen with Galpha-s. The disappearance and reappearance of Galpha-i and Gbeta-gamma subunits from and to the detergent-soluble fractions occur with similar time courses as observed for the onset and disappearance of AC superactivation. Lastly, pertussis toxin, which blocks acute and chronic agonist-induced AC inhibition and superactivation, also blocks the shift in detergent solubility. These results suggest a correlation between the solubility shift of the heterotrimeric Gi protein and the generation of AC superactivation.

144. Rhee M.-H., Nevo I., Avidor-Reiss T., Levy R., and Vogel Z. (2000) Differential superactivation of adenylyl cyclase isozymes following chronic activation of the CB1 cannabinoid receptor. Mol. Pharmacol. 57:746-752.
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Abstract: Many types of cells exhibit increased adenylyl cyclase (AC) activity after chronic agonist treatment of Gi/o-coupled receptors. This phenomenon, defined as AC superactivation or sensitization, has mostly been studied for the opioid receptors and is implicated in opiate addiction. Here we show that this phenomenon is also observed on chronic activation of the CB1 cannabinoid receptor. Moreover, using COS-7 cells cotransfected with CB1 receptor and individual AC isozymes, we could show selective superactivation of AC types I, III, V, VI, and VIII. The level of superactivation was dependent on the concentration of agonist and time of agonist exposure and was not dependent on the AC stimulator used. No superactivation of AC types II, IV, or VII was observed in COS-7 cells cotransfected with CB1. The superactivation of AC type V was abolished by pretreatment with pertussis toxin and by cotransfection with the carboxy terminus of beta-adrenergic receptor kinase, which serves as a scavenger of Gbeta-gamma dimers, implying a role for the Gi/o proteins and especially Gbeta-gamma dimers in the cannabinoid-induced superactivation of AC.

145. Eckhardt K., Nevo I., Levy R., Mikus G., Eichelbaum M., and Vogel Z. (2000) Morphine-related metabolites differentially activate adenylyl cyclase isozymes after acute and chronic administration. FEBS Lett. 470:309-314.
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Abstract: Morphine-3- and morphine-6-glucuronide are morphine's major metabolites. As morphine-6-glucuronide produces stronger analgesia than morphine, we investigated the effects of acute and chronic morphine glucuronides on adenylyl cyclase (AC) activity. Using COS-7 cells cotransfected with representatives of the nine cloned AC isozymes, we show that AC-I and V are inhibited by acute morphine and morphine-6-glucuronide, and undergo superactivation upon chronic exposure, while AC-II is stimulated by acute and inhibited by chronic treatment. Morphine-3-glucuronide had no effect. The weak opiate agonists codeine and dihydrocodeine are also addictive. These opiates, in contrast to their 3-O-demethylated metabolites morphine and dihydromorphine (formed by cytochrome P450 2D6), demonstrated neither acute inhibition nor chronic-induced superactivation. These results suggest that metabolites of morphine (morphine-6-glucuronide) and codeine/dihydrocodeine (morphine/dihydromorphine) may contribute to the development of opiate addiction.

146. Shapira M., Vogel Z., and Sarne Y. (2000) Opioid and cannabinoid receptors share a common pool of GTP-binding proteins in cotransfected cells, but not in cells which endogenously coexpress the receptors. Cell. Mol. Neurobiol. 20:291-304.

Abstract:1. Opioid (mu. delta, kappa) and cannabinoid (CB1, CB2) receptors are coupled mainly to G(i)/G(o) GTP-binding proteins. The goal of the present study was to determine whether different subtypes of opioid and cannabinoid receptors, when coexpressed in the same cell, share a common reservoir. or utilize different pools, of G proteins. 2. The stimulation of [S-35]GTP gamma S binding by selective opioid and cannabinoid agonists was tested in transiently transfected COS-7 cells, as well as in neuroblastoma cell lines. In COS-7 cells, cotransfection of mu- and delta-opioid receptors led to stimulation of [S-35]GTP gamma S binding by either mu-selective (DAMGO) or delta-selective (DPDPE) agonists. The combined effect of the two agonists was similar to the effect of either DAMGO or DPDPE alone, suggesting the activation of a common G-protein reservoir by the two receptor subtypes. 3. The same phenomenon was observed when COS-7 cells were cotransfected with CB1 cannabinoid receptors and either mu- or delta-opioid receptors. 4. On the other hand. in N18STG2 neuroblastoma cells, which endogenously coexpress CB1 and delta-opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress mu- and delta-opioid receptors, the combined effects of the various agonists (the selective cannabinoid DALN and the selective opioids DPDPE and DAMGO) were additive, implying the activation of different pools of G proteins by each receptor subtype. 5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.

147. Rhee M.-H., Nevo I., Bayewitch M.L., Zagoory O., and Vogel Z. (2000) Functional role of tryptophan residues in the fourth transmembrane domain of the CB2 cannabinoid receptor. J. Neurochem. 75:2485-2491.
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Abstract: Several tryptophan (Trp) residues are conserved in G protein-coupled receptors (GPCRs). Relatively little is known about the contribution of these residues, and especially of those in the 4th transmembrane domain, in the function of the CB2 cannabinoid receptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conserved in CB1 and CB2 cannabinoid receptors, but not in many other GPCRs), of the human CB2 receptor, with A or L, or with F or Y, produced different results. We found that the conservative change of W172 to F or Y retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase), whereas removal of the aromatic side chain by mutating W172 to A or L eliminated agonist binding. W158 was even more sensitive to being mutated. We found that the conservative W158F mutation retained wild type binding and signaling activities. However, W158Y and W158A mutants completely lost ligand binding capacity. Thus, the Trp side chains at positions 158 and 172 seem to have a critical, but different, role in cannabinoid binding to the human CB2 receptor.

148. Hanus L., Abu-Lafi S., Fride E., Breuer A., Vogel Z., Shalev D.E., Kustanovich I., and Mechoulam R. (2001) 2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB1 receptor. Proc. Natl. Acad. Sci. 98:3662-3665.
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Abstract: Two types of endogenous cannabinoid-receptor agonists have been identified thus far. They are the ethanolamides of polyunsaturated fatty acids-arachidonoyl ethanolamide (anandamide) is the best known compound in the amide series-and 2-arachidonoyl glycerol, the only known endocannabinoid in the ester series. We report now an example of a third, ether-type endocannabinoid, 2-arachidonyl glyceryl ether (noladin ether), isolated from porcine brain. The structure of noladin ether was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by comparison with a synthetic sample. It binds to the CB1 cannabinoid receptor (Ki = 21.2 +/- 0.5 nM) and causes sedation, hypothermia, intestinal immobility, and mild antinociception in mice. It binds weakly to the CB2 receptor (Ki > 3 mM).

149. Scarselli M., Novi F., Schallmach E., Lin R., Baragli A., Colzi A., Griffon N., Corsini G.U., Sokoloff P., Levenson R., Vogel Z., and Maggio R. (2001) D2/D3 dopamine receptor heterodimers exhibit unique functional properties. J. Biol. Chem. 276:30308-30314.
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Abstract: Evidence for heterodimerization has recently been provided for dopamine D1 and adenosine A1 receptors as well as for dopamine D2 and somatostatin SSTR5 receptors. In this paper, we have studied the possibility that D2 and D3 receptors interact functionally by forming receptor heterodimers. Initially, we split the two receptors at the level of the third cytoplasmic loop into two fragments. The first, containing transmembrane domains (TM) I to V and the N-terminal part of the third cytoplasmic loop, was named D2trunk or D3trunk, and the second, containing the C-terminal part of the third cytoplasmic loop, TMVI and TMVII, and the C-terminal tail, was named D2tail or D3tail. Then we defined the pharmacological profiles of the homologous (D2trunk/D2tail and D3trunk/D3tail) as well as of the heterologous (D2trunk/D3tail and D3trunk/D2tail) cotransfected receptor fragments. The pharmacological profile of the cross-cotransfected fragments was different from that of the native D2 or D3 receptors. In most cases, the D3trunk/D2tail was the one with the highest affinity for most agonists and antagonists. Moreover, we observed that all of these receptor fragments reduced the expression of the wild type dopamine D2 and D3 receptors, suggesting that D2 and D3 receptors can form complexes with these fragments and that these complexes bind [3H]nemonapride less efficiently or are not correctly targeted to the membrane. In a second set of experiments, we tested the ability of the split and the wild type receptors to inhibit adenylyl cyclase (AC) types V and VI. All of the native and split receptors inhibited AC-V and AC-VI, with the exception of D3, which was unable to inhibit AC-VI. We therefore studied the ability of D2 and D3 to interact functionally with one another to inhibit AC-VI. We found that with D2 alone, R-(+)-7-hydroxydypropylaminotetralin hydrobromide inhibited AC-VI with an IC50 of 2.05 +/- 0.15 nM, while in the presence of D2 and D3 it inhibited AC-VI with an IC50 of 0.083 +/- 0.011 nM. Similar results were obtained with a chimeric cyclase made from AC-V and AC-VI. Coimmunoprecipitation experiments indicate that D2 and D3 receptors are capable of physical interaction.

150. Di Marzo V., Melck D., Orlando P., Bisogno T., Zagoory O., Bifulco M., Vogel Z., and De Petrocellis L. (2001) Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells. Biochem. J. 358:249-255.
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Abstract: Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an endogenous agonist of cannabinoid receptor type 1 (CB1). This synergistic effect was reduced by the CB2 cannabinoid receptor antagonist SR144528, although PEA does not activate either CB1 or CB2 receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (1-10 uM) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5 uM) decreased the IC50 values for AEA inhibitory effects by 3-6-fold. This effect was not blocked by the CB2 receptor antagonist SR144528, and was not mimicked by a selective agonist of CB2 receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5 uM PEA caused a ca. 30-40% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB1 or CB2 receptors, and neither did it alter the CB1/CB2-mediated inhibitory effect of AEA on adenylate cyclase type V, nor the expression of CB1 and CB2 receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression.