Biomedical subjects

Z Vogel

Publications and source records attributed to Z Vogel.

At least 19 recordsLinked to original sources

PubMed2005

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.

Animals↗

PubMed2001

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.

Amides↗

PubMed2001

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.

Adenylyl Cyclases↗

PubMed2001

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).

Animals↗

PubMed2000

Morphine-related metabolites differentially activate adenylyl cyclase isozymes after acute and chronic administration.

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.

Adenylyl Cyclase Inhibitors↗

PubMed2000

Role of the highly conserved Asp-Arg-Tyr motif in signal transduction of the CB2 cannabinoid receptor.

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.

Amino Acid Motifs↗

PubMed2000

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 Cyclases↗

PubMed2000

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.

Analgesics↗

PubMed2000

Functional role of tryptophan residues in the fourth transmembrane domain of the CB(2) cannabinoid receptor.

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 fourth transmembrane domain in the function of the CB(2) cannabinoid receptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conserved in CB(1) and CB(2) cannabinoid receptors but not in many other GPCRs) of the human CB(2) 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 CB(2) receptor.

Amino Acid Substitution↗

PubMed2000

Differential superactivation of adenylyl cyclase isozymes after chronic activation of the CB(1) cannabinoid receptor.

Many types of cells exhibit increased adenylyl cyclase (AC) activity after chronic agonist treatment of G(i/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 CB(1) cannabinoid receptor. Moreover, using COS-7 cells cotransfected with CB(1) 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 CB(1). 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 G(betagamma) dimers, implying a role for the G(i/o) proteins and especially G(betagamma) dimers in the cannabinoid-induced superactivation of AC.

Adenylyl Cyclases↗

PubMed2000

Alterations in detergent solubility of heterotrimeric G proteins after chronic activation of G(i/o)-coupled receptors: changes in detergent solubility are in correlation with onset of adenylyl cyclase superactivation.

Prolonged G(i/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 G(i)-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 G(i/o)-coupled receptors (i.e., mu- and kappa-opioid, and m(4)-muscarinic), we observed that although no overall change occurred in total content of G(alphai)- and beta(1)-subunits, chronic agonist treatment led to a marked reduction in the ability of 1% cholate to solubilize G(betagamma) as well as G(alphai). This solubility shift is exclusively observed with G(alphai), and was not seen with G(alphas). The disappearance and reappearance of G(alphai) and G(betagamma) 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 G(i) protein and the generation of AC superactivation.

Adenylate Cyclase Toxin↗

PubMed1999

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.

Adenylate Cyclase Toxin↗

PubMed1998

An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity.

2-Arachidonoyl-glycerol (2-Ara-GI) 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-GI is accompanied by several 2-acyl-glycerol esters, two major ones being 2-linoleoyl-glycerol (2-Lino-Gl) and 2-palmitoyl-glycerol (2-Palm-Gl). These two esters 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-Gl and its apparent capacity to inhibit adenylyl cyclase. Together these esters also significantly potentiate 2-Ara-Gl inhibition of motor behavior, immobility on a ring, analgesia on a hot plate and hypothermia caused by 2-Ara-Gl in mice. 2-Lino-Gl, but not 2-Palm-GI, significantly inhibits the inactivation of 2-Ara-Gl by neuronal and basophilic cells. These data indicate that the biological activity of 2-Ara-Gl 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.

Adenylyl Cyclase Inhibitors↗

PubMed1998

Differential modulation of adenylyl cyclases I and II by various G beta subunits.

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 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 and inhibited by G beta 5/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.

Adenylyl Cyclases↗

PubMed1998

Interaction of agonist peptide [3H]Tyr-D-Ala-Phe-Phe-NH2 with mu-opioid receptor in rat brain and CHO-mu/1 cell line.

Opioid receptor binding properties of [3H]Tyr-D-Ala-Phe-Phe-NH2 (TAPP) were characterized in rat brain and Chinese hamster ovary (CHO) cells expressing the rat mu-receptor. In rat brain, [3H]TAPP labeled a single class of opioid sites with a dissociation constant (Kd) of 0.31 nM and maximal number of binding sites (Bmax) of 119 fmol/mg protein. In CHO-mu/1 cell membranes, the Kd and Bmax 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/1 cell membranes and modulated by Na+ ions and guanine nucleotides. The high affinity and selectivity of [3H]TAPP together with its low non-specific binding make this radioligand a useful tool for labeling the native and cloned mu-opioid receptor.

Animals↗

PubMed1998

Nociceptive stimulus induces release of endogenous beta-endorphin in the rat brain.

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.

Animals↗

PubMed1998

Opioid modulation of extracellular signal-regulated protein kinase activity is ras-dependent and involves Gbetagamma subunits.

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-Ala2,Me-Phe4,Gly-ol5]enkephalin)-, delta- ([D-Pen2,D-Pen5]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 betagamma 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 betagamma 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.

Animals↗

PubMed1998

Cannabinoid receptor activation differentially regulates the various adenylyl cyclase isozymes.

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 betagamma 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.

Adenylyl Cyclase Inhibitors↗