Michael Bayewitch

Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors.

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 Ki 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.

The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling

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 Gi/G0 GTP‐binding proteins. Interestingly, contrary to results with CB1, anandamide did not inhibit the CB2 coupled adenylate cyclase activity and δ 9‐tetrahydrocannabinol had only marginal effects. These results characterize the CB2 receptor as a functional and distinctive member of the cannabinoid receptor family.

OPIATE-INDUCED ADENYLYL CYCLASE SUPERACTIVATION IS ISOZYME-SPECIFIC

While acute activation of inhibitory Gi/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 μ-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 μ-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.

Cannabinoid receptor activation differentially regulates the various adenylyl cyclase isozymes.

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 Gi/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 αs, αi/o, and βγ 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 αs, 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 Interaction with the D2 Dopamine Receptor Family; Differential Coupling to Gi, Gz, and Gs

Abstract1.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 α subunit of Gz. PTX treatment was employed to inactivate endogenous αi, 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.

Differential Modulation of Adenylyl Cyclases I and II by Various Gβ Subunits

The accepted dogma concerning the regulation of adenylyl cyclase (AC) activity by Gβγ dimers states that the various isoforms of AC respond differently to the presence of free Gβγ. It has been demonstrated that AC I activity is inhibited and AC II activity is stimulated by Gβγ subunits. This result does not address the possible differences in modulation that may exist among the different Gβγ heterodimers. Six isoforms of Gβ and 12 isoforms of Gγ have been cloned to date. We have established a cell transfection system in which Gβ and Gγ 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 Gβ1/γ2 and Gβ5/γ2 combinations, AC II responded differentially and was stimulated by Gβ1/γ2 and inhibited by Gβ5/γ2. This finding demonstrates differential modulatory activity by different combinations of Gβγ on the same AC isoform and demonstrates another level of complexity within the AC signaling system.

Inhibition of adenylyl cyclase isoforms V and VI by various Gβγ subunits

An intriguing development in the G‐protein signaling field has been the finding that not only the Gα subunit, but also Gβγ subunits, affect a number of downstream target molecules. One of the downstream targets of Gβγ is adenylyl cyclase, and it has been demonstrated that a number of isoforms of adenylyl cyclase can be either inhibited or stimulated by Gβγ subunits. Until now, adenylyl cyclase type I has been the only isoform reported to be inhibited by free Gβγ. Here we show by transient cotransfection into COS‐7 cells of either adenylyl cyclase V or VI, together with Gγ2 and various Gβ subunits, that these two adenylyl cyclase isozymes are markedly inhibited by Gβγ. In addition, we show that Gβ1 and Gβ5 subunits differ in their activity. Gβ1 transfected alone markedly inhibited adenylyl cylcase V and VI (probably by recruiting endogenous Gγ subunits). On the other hand, Gβ5 produced less inhibition of these isozymes, and its activity was enhanced by the addition of Gγ2. These results demonstrate that adenylyl cyclase types V and VI are inhibited by Gβγ dimers and that Gβ1 and Gβ5 subunits differ in their capacity to regulate these adenylyl cyclase isozymes.—Bayewitch, M. L., Avidor‐Reiss, T., Levy, R., Pfeuffer, T., Nevo, I., Simonds, W. F., Vogel, Z. Inhibition of adenylyl cyclase isoforms V and VI by various Gβγ subunits. FASEB J. 12, 1019–1025 (1998)

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.

ENDOGENOUS CANNABINOID LIGANDS : CHEMICAL AND BIOLOGICAL STUDIES

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.

Functional Role of Tryptophan Residues in the Fourth Transmembrane Domainof the CB2 Cannabinoid Receptor

Abstract: Several tryptophan (Trp) residues are conserved in Gprotein‐coupled receptors (GPCRs). Relatively little is known about thecontribution of these residues and especially of those in the fourthtransmembrane domain in the function of the CB2 cannabinoidreceptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conservedin CB1 and CB2 cannabinoid receptors but not in manyother GPCRs) of the human CB2 receptor with A or L or with F or Yproduced different results. We found that the conservative change of W172 to For Y retained cannabinoid binding and downstream signaling (inhibition ofadenylyl cyclase), whereas removal of the aromatic side chain by mutating W172to A or L eliminated agonist binding. W158 was even more sensitive to beingmutated. We found that the conservative W158F mutation retained wild‐typebinding and signaling activities. However, W158Y and W158A mutants completelylost ligand binding capacity. Thus, the Trp side chains at positions 158 and172 seem to have a critical, but different, role in cannabinoid binding to thehuman CB2 receptor.