| Neurobiology Department
Weizmann Institute of Science
Prof. Zvi Vogel's laboratory
Drug abuse is one of the most notorious socioeconomic problems of our time. Although this phenomenon has existed for a long time, it has recently acquired epidemic proportions worldwide. The abuse of cannabinoids (considered "soft drugs") is one of the most prevalant, but leads the user to the abuse of "harder" drugs. In this regard, the abuse of opiates (i.e. heroin, methadone) constitutes the most dangerous and noxious kind of drug addiction. In addition to the direct human affliction, the use of drugs of abuse is a major factor in urban criminality and in the spread of infectious diseases, including AIDS. Moreover, both opiates and cannabinoids have important beneficial medical properties. Cannabinoids have analgesic and antiemetic properties, while opiates are known as the most effective analgesic (pain-alleviating) medications (e.g. morphine in surgery) and should serve as drugs of choice for the relief of chronic or acute pain. However, the use of such drugs is currently legally restricted (in most countries) due to their addictive properties, thus limiting their medical use.A. Regulation of adenylyl cyclase by acute and chronic opiate exposure: Relevance to opiate addiction
Very little is known about the physiological and molecular mechanisms by which opiates function. Even less is known about the biochemical changes occurring upon chronic opiate exposure, exposure that leads to the development of opiate tolerance and dependence. Furthermore, no information is available regarding the mechanisms that accompany the subsequent phase of opiate withdrawal, a phenomenon which causes suffering to the abuser and is the major reason for his reluctance to give up the use of drugs. Our long-term goals are to elucidate the mechanisms involved in opiate actions, and to characterize the biochemical changes accompanying chronic opiate treatment and the withdrawal phase.
We have recently identified and characterized several important molecules which are affected by opiate treatment and have a key function in transmitting signals through the cell membrane. Most of our recent work was centered on the characterization of the modulation by opiates of the various types of the enzyme adenylyl cyclase (AC). Using Chinese hamster ovary (CHO) cells transfected with m or k opiate receptors, we were able to show that while acute exposure to opiate agonists leads to AC inhibition, chronic exposure results in AC superactivation. This activation of AC by chronic opiates (particularly evident upon withdrawal of the inhibitory opiate agonist) has been defined as AC superactivation or sensitization, and seems to play an important role in opiate addiction and the withdrawal phenomenon. Nine types of AC isozymes are currently known. These isozymes differ in their tissue distribution and in their stimulation/inhibition patterns. It was therefore of interest to determine the pattern of regulation of the various AC isozymes by acute and chronic opiate treatments.
Utilizing COS-7 cells transfected with cDNAs encoding the m-opiate receptor and the individual AC isozymes (types IVIII), we concluded that the population of AC isozymes could be divided into three groups: (i) AC-I, V, VI and VIII are inhibited by acute opiate treatment and superactivated by chronic exposure, with AC-V (known to be localized to brain areas involved in drug addiction and reward mechanisms) yielding the largest superactivation; (ii) AC-II, IV and VII are stimulated by acute opiate exposure and do not show superactivation; and (iii) AC-III seems not to be significantly affected by either acute or chronic opiate exposure. We have also shown that the phenomenon of AC superactivation by chronic agonist exposure is of a general nature, and that AC-I, V, VI and VIII are also superactivated following chronic activation by other Gi/o-coupled receptors (e.g. CB1 cannabinoid, D2-dopaminergic, and m2- and m4-muscarinic).
The second step of this research work involved investigating the role of Gbg dimers (known to be released from Gai/o upon receptor activation) in AC regulation. Using transfected COS cells, we confirmed that AC-I is inhibited and AC-II is stimulated by Gb1g2. However, we found that different Gb subunits differ in their capacity to regulate AC activity, with Gb1 stimulating AC-II, and Gb5 (a Gb isoform found at high concentrations in the brain) inhibiting this AC isozyme. In a similar series of experiments, we found that Gb1 inhibits the activities of AC-V and VI (a much weaker inhibition was observed with Gb5).
Moreover, Gbg scavengers (i.e. molecules which bind Gbg subunits and interfere with its modulation of AC activity) increased the activity of AC-V and AC-VI and prevented this superactivation by chronic opiate agonist exposure. This study suggests that endogenous Gbg tonically inhibits the activity of AC-V (and of AC-VI), and that this tonic inhibition is reversed by the chronic agonist exposure. We are currently studying whether the effect of Gbg on AC-V and VI is direct or indirect. In this regard, in a recent study we showed that anti-Gb1 antibody co-immunoprecipitated AC-V, suggesting a direct interaction between the two molecules. We are currently investigating the interaction of Gb1 with the C1 and C2 intracellular domains of AC-V. We found that Gbg interacts with the C1 loop, and have localized such a binding area to a fragment of 65 amino acids (fragment 445-509 in C1). A specific mutation in this area abolished Gbg binding to the fragment. Moreover, when the same mutation was introduced into the AC-V molecule, its capacity to be superactivated was markedly reduced, suggesting that Gbg binding to this area of AC is important for the superactivation phenomenon.
Future Plans: Employing cultured cells and molecular biological techniques, we plan to continue to reveal the molecular mechanisms by which opiates and agonists of other Gi/o-coupled receptors affect the activity of AC and to determine how changes in AC and other signaling molecules may underlie the physiological phenomenon of opiate drug addiction. These studies should advance our understanding of opiate function under normal, tolerance and withdrawal conditions as well as increase our knowledge of the molecular events involved in opiate control of neuronal communication. In addition, the results of such studies may provide tools that should eventually contribute to new clinical strategies in the prevention and reversal of drug addiction.B. Properties of endogenous ligands to cannabinoid receptors
A few years ago, it was shown that the brain, as well as several peripheral tissues, have receptors which interact with the active material (D9-THC) present in cannabis (marijuana, hashish), and which are responsible for mediating the psychotropic activities of this material. In collaboration with Raphael Mechoulam (Hebrew University, Jerusalem), we searched for endogenous materials in brain and other tissues which interact with these receptors. This search was based on the belief that if D9-THC acts on such receptors, then there should be some endogenous compounds that regulate the activity of these receptors. Indeed, this search led to the discovery of two families of endogenous cannabinoid ligands, including that of anandamide (arachidonoyl-ethanolamide) and that of 2-arachidonoyl-glycerol. We found that these two types of materials interact with the "brain cannabinoid receptor" (CB1), located in neural cells and tissues, and to a lesser extent with the "peripheral cannabinoid receptor" (CB2), expressed in various cells of the immune system, and induce the proper physiological responses known to be evoked by the "classical" tricyclic cannabinoids. This discovery has opened the field of cannabinoids, allowing many groups to find roles for these materials in activities as diverse as reproduction, immune response, control of blood pressure, and cell proliferation, in addition to the known psycotropic effects of these endogenous materials.
More recently, we assayed various derivatives of the endogenous ligands, as well as of the tricyclic cannabinoids, for their biological and pharmacological properties. We also found that the endogenous cannabinoid ligands are present in body tissues together with numerous additional pharmacologically inactive (or partially active) acylamides and 2-acyl-glycerol esters. We have established that these "entourage compounds" enhance receptor binding of the endogenous ligands, stabilize them against hydrolysis, and potentiate their in vivo and in vitro effects. We are currently screening these materials for their biological and therapeutic effects.
Future Plans: The characterization of various derivatives of endogenous and tricyclic cannabinoids should expand our understanding of the specific functions of the brain and peripheral cannabinoid receptors, and open new pathways in the development of receptor-selective novel cannabinoid drugs, such as anti-inflammatory agents, drugs for treatment of glaucoma, and compounds which exert behavioral effects.