Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death.

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.

Delta9-tetrahydrocannabinol increases C6 glioma cell death produced by oxidative stress.

(-)Delta9-tetrahydrocannabinol is a scavenger of free radicals. However, the activation of the CB1 receptor in cultured C6 glioma cells by (-)delta9-tetrahydrocannabinol in the presence of reagents generating reactive oxygen species leads to amplification of the cellular damage from oxidative stress. This was evident by increased loss of cell wall integrity, impaired mitochondrial function and reduction of glucose uptake. In addition, (-)delta9-tetrahydrocannabinol treatment was also found to be deleterious to the cells under conditions of glucose starvation. Free radicals have been implicated in various conditions leading to cell death and, as a routine, the Fenton reaction is utilized for modeling reactive oxygen species production. Our study was performed using a cell permeating Fe(III) chelating quinone that provides more physiological conditions for mimicking the naturally occurring oxidative stress within the cell and thus serves as a better model for natural reactive oxygen species formation.

Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells.

Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an endogenous agonist of cannabinoid receptor type 1 (CB(1)). This synergistic effect was reduced by the CB(2) cannabinoid receptor antagonist SR144528, although PEA does not activate either CB(1) or CB(2) 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 microM) 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 microM) decreased the IC(50) values for AEA inhibitory effects by 3-6-fold. This effect was not blocked by the CB(2) receptor antagonist SR144528, and was not mimicked by a selective agonist of CB(2) 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 microM PEA caused a approximately 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 CB(1) or CB(2) receptors, and neither did it alter the CB(1)/CB(2)-mediated inhibitory effect of AEA on adenylate cyclase type V, nor the expression of CB(1) and CB(2) 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.

D2/D3 dopamine receptor heterodimers exhibit unique functional properties.

Evidence for heterodimerization has recently been provided for dopamine D(1) and adenosine A(1) receptors as well as for dopamine D(2) and somatostatin SSTR(5) receptors. In this paper, we have studied the possibility that D(2) and D(3) 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 D(2trunk) or D(3trunk), and the second, containing the C-terminal part of the third cytoplasmic loop, TMVI and TMVII, and the C-terminal tail, was named D(2tail) or D(3tail). Then we defined the pharmacological profiles of the homologous (D(2trunk)/D(2tail) and D(3trunk)/D(3tail)) as well as of the heterologous (D(2trunk)/D(3tail) and D(3trunk)/D(2tail)) cotransfected receptor fragments. The pharmacological profile of the cross-cotransfected fragments was different from that of the native D(2) or D(3) receptors. In most cases, the D(3trunk)/D(2tail) 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 D(2) and D(3) receptors, suggesting that D(2) and D(3) receptors can form complexes with these fragments and that these complexes bind [(3)H]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 D(3), which was unable to inhibit AC-VI. We therefore studied the ability of D(2) and D(3) to interact functionally with one another to inhibit AC-VI. We found that with D(2) alone, R-(+)-7-hydroxydypropylaminotetralin hydrobromide inhibited AC-VI with an IC(50) of 2.05 +/- 0.15 nm, while in the presence of D(2) and D(3) it inhibited AC-VI with an IC(50) 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 D(2) and D(3) receptors are capable of physical interaction.

2-arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB1 receptor.

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 CB(1) cannabinoid receptor (K(i) = 21.2 +/- 0.5 nM) and causes sedation, hypothermia, intestinal immobility, and mild antinociception in mice. It binds weakly to the CB(2) receptor (K(i) > 3 microM).

Opioid and cannabinoid receptors share a common pool of GTP-binding proteins in cotransfected cells, but not in cells which endogenously coexpress the receptors.

1. Opioid (mu, delta, kappa) and cannabinoid (CB1, CB2) receptors are coupled mainly to Gi/Go 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 [35S]GTPgammaS 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 [35S]GTPgammaS 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 N18TG2 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.

Acute and chronic activation of the mu-opioid receptor with the endogenous ligand endomorphin differentially regulates adenylyl cyclase isozymes.

While acute activation of G(i/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.

Adenylyl cyclase interaction with the D2 dopamine receptor family; differential coupling to Gi, Gz, and Gs.

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.

Regulation of adenylyl cyclase isozymes on acute and chronic activation of inhibitory receptors.

Adenylyl cyclase superactivation, a phenomenon by which chronic activation of inhibitory Gi/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 D2 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.

(-)-Delta9-tetrahydrocannabinol antagonizes the peripheral cannabinoid receptor-mediated inhibition of adenylyl cyclase.

(-)-Delta9-Tetrahydrocannabinol ((-)-Delta9-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 (-)-Delta9-THC binds to both receptors with similar affinity. However, in contrast to its capacity to serve as an agonist for the CB1 receptor, (-)-Delta9-THC was only able to induce a very slight inhibition of adenylyl cyclase at the CB2 receptor. Morever, (-)-Delta9-THC antagonizes the agonist-induced inhibition of adenylyl cyclase mediated by CB2. Therefore, we conclude that (-)-Delta9-THC constitutes a weak antagonist for the CB2 receptor.

Correlation between secretagogue-induced Ca2+ influx, intracellular Ca2+ levels and secretion of catecholamines in cultured adrenal chromaffin cells.

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 microM) 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.

M1 agonists for the treatment of Alzheimer's disease. Novel properties and clinical update.

The AF series compounds, AF102B and congeners of AF150(S), are functionally selective agonists for m1 muscarinic receptors (m1AChRs). This is shown in stable transfected CHO and PC12 cells (PC12M1) with m1m5AChRs and m1AChRs, 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 AF 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 m1AChRs 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 m1AChR in PC12M1 cells with carbachol or AF102B decreases tau phosphorylation as indicated by specific tau-1 mAb and alkaline phosphatase treatment. Due to the above mentioned properties m1 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 m1 agonists. These include studies on AF102B and xanomeline, another m1 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.

Adenylylcyclase supersensitization in mu-opioid receptor-transfected Chinese hamster ovary cells following chronic opioid treatment.

Using CHO cells stably transfected with rat mu-opioid receptor cDNA, we show that the mu-agonists morphine and [D-Ala2,N-methyl-Phe4,Gly-ol5]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-Ala2,N-methyl-Phe4,Gly-ol5]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 Gi/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.

Cannabinomimetic behavioral effects of and adenylate cyclase inhibition by two new endogenous anandamides.

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, docosatetraenylethanolamide (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 K1 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 E1-stimulated adenylate cyclase activity in Chinese hamster ovary-K1 cells transfected with the cannabinoid receptor, as well as in N18TG2 mouse neuroblastoma cells that express the cannabinoid receptor naturally. The IC50 values for the inhibition of adenylate cyclase in transfected Chinese hamster ovary-K1 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 delta9-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 delta9-tetrahydrocannabinol and arachidonylethanolamide: inhibition of motor activity in an open field, hypothermia, catalepsy on a ring, and analgesia on a hot plate.

kappa-Opioid receptor-transfected cell lines: modulation of adenylyl cyclase activity following acute and chronic opioid treatments.

The opioid receptors mu, delta and kappa have recently been cloned. Here we 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 Gi/Go proteins in both processes.

Low doses of anandamides inhibit pharmacological effects of delta 9-tetrahydrocannabinol.

It has been shown previously that the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide 20:4, n-6) induces in vivo and in vivo effects typical of a cannabinoid partial agonist. We now report that the synthetic docosahexaenylethanolamide (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.

The fourth immunoglobulin domain of the stem cell factor receptor couples ligand binding to signal transduction.

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.

Opioids inhibit endothelin-mediated DNA synthesis, phosphoinositide turnover, and Ca2+ mobilization in rat C6 glioma cells.

Opioid agonists inhibit DNA synthesis in C6 rat glioma cells that express opioid receptors, induced by desipramine (DMI). This inhibition was not observed in cells that were not treated with DMI, and thus did not express opioid-binding sites. Endothelin, a known mitogen, increased thymidine incorporation dose dependently (up to 1.7-fold) in DMI-treated C6 cells. This increase was reversed by an anti-idiotypic antibody to opioid receptors, Ab2AOR, which has opioid agonist properties. The opioid antagonist naltrexone blocked the inhibition caused by Ab2AOR. Endothelin also stimulated phosphoinositide (PI) turnover and this effect was inhibited by morphine (50%) or by Ab2AOR (72%) in DMI-treated but not in DMI-untreated C6 cells. These actions of morphine and Ab2AOR were reversed by naltrexone. The inhibition of PI turnover and of thymidine incorporation by Ab2AOR or morphine was insensitive to pertussis toxin (PTX). Since PI turnover is known to induce Ca2+ mobilization, it was of interest to examine the effects of the applied opioids on intracellular Ca2+ concentrations. Endothelin increased the concentration of cytosolic free Ca2+ in the cells while Ab2AOR, morphine, and beta-endorphin reversed the endothelin-induced Ca2+ mobilization in DMI-treated but not in DMI-untreated C6 cells. The effect of these agonists was also blocked by naltrexone. The results indicate that glial cells can be a target of an opioid receptor-mediated antimitogenic action and that an abatement in PI turnover and Ca2+ mobilization may be associated with this mechanism.

A monoclonal anti-idiotypic antibody to opioid receptors labels desipramine-induced opioid binding sites on rat C6 glioma cells and attenuates thymidine incorporation into DNA.

Treatment of rat C6 glioma cells with the tricyclic antidepressant desipramine induces opioid binding. Here the distribution of these opioid-binding sites on C6 cell membranes and a functional property were investigated. Immunohistochemical examination of C6 cells was performed using a monoclonal anti-idiotypic antibody to opioid receptors (Ab2AOR). Ab2AOR uniformly labeled > 97% of the cells exposed to desipramine over their entire surface. The opioid-receptor antagonist naltrexone completely blocked Ab2AOR binding. Ab2AOR, which has opioid agonist properties, also inhibited DNA synthesis in desipramine-treated but not in naive C6 cells. Similarly, morphine blocked C6 cell proliferation only after desipramine treatment. The antineurotrophic action of Ab2AOR was reversed by naltrexone and was insensitive to pertussis toxin. These findings demonstrate that Ab2AOR suppresses the proliferation of C6 glioma cells by binding to desipramine-induced opioid receptors.

Anandamide, a brain endogenous compound, interacts specifically with cannabinoid receptors and inhibits adenylate cyclase.

A putative endogenous cannabinoid ligand, arachidonylethanolamide (termed "anandamide"), was isolated recently from porcine brain. Here we demonstrate that this compound is a specific cannabinoid agonist and exerts its action directly via the cannabinoid receptors. Anandamide specifically binds to membranes from cells transiently (COS) or stably (Chinese hamster ovary) transfected with an expression plasmid carrying the cannabinoid receptor DNA but not to membranes from control nontransfected cells. Moreover, anandamide inhibited the forskolin-stimulated adenylate cyclase in the transfected cells and in cells that naturally express cannabinoid receptors (N18TG2 neuroblastoma) but not in control nontransfected cells. As with exogenous cannabinoids, the inhibition by anandamide of the forskolin-stimulated adenylate cyclase was blocked by treatment with pertussis toxin. These data indicate that anandamide is an endogenous agonist that may serve as a genuine neurotransmitter for the cannabinoid receptor.