Signal Transduction and Addiction: The Role of Ion Channels and Adenylyl Cyclase
Our laboratory is focused on delineating the biological factors which contribute to alcohol and other drug addictions. We concentrate especially on two major signal transduction systems - the NMDA receptor-gated ion channels and adenylyl cyclase. Our goal is to elucidate the role of these systems in both the acute actions of sedative hypnotic drugs, and the neuroadaptation processes that result in tolerance to, and physical dependence on, these drugs. Interest in these signal transduction systems was spurred by our conceptualization of alcohol tolerance and physical dependence as examples of biological memory processes, and by the known involvement of the NMDA receptor and adenylyl cyclase systems in various forms of memory.
In our research, we have used calcium imaging to investigate acute and chronic actions of ethanol on the NMDA receptor system in cultured neurons, and receptor autoradiography to access changes in NMDA receptor systems in brains of ethanol-dependent animals. We were the initial group to show the very potent inhibition by ethanol of calcium influx through the NMDA receptor-gated ion channel, and have proceeded to demonstrate the importance of PKC mediated phosphorylation mechanisms and co-agonist (glycine) affinity at the NMDA receptor in ethanol's acute actions. Currently, our work demonstrates that the acute actions of ethanol are a result of a phosphorylation-dependent shift in glycine affinity which involves a subgroup of brain NMDA receptors. The consequence of our findings is that glycine site agonists can be considered as amethystine agents which may be used to reverse some portion of ethanol's acute intoxicating effects.
Our work has also shown that chronic exposure of both cultured neurons and the brains of live animals to ethanol results in an up-regulation of NMDA receptors. This event is consonant with an increase in neural excitability, manifesting itself as seizures during ethanol withdrawal and sensitizing neurons to withdrawal-induced excitotoxicity and cell death. When we fed mice with ethanol-containing liquid diets for a period of time known to generate physical dependence on ethanol in these animals, we found that the increase in brain NMDA receptors was evident over a time course which mirrored the time course for the susceptibility to withdrawal-induced convulsions. Using primary cultures of cellebular granule cells, we found that growing these cells in media containing ethanol produced both increases in NMDA receptor number and increases in NMDA receptor function in these cultured cells. If the ethanol-treated cells were exposed to glutamate after the ethanol treatment, significantly more cells underwent necrosis in the ethanol-treated/withdrawn population of cells compared to the control (non-ethanol treated) population. We have further demonstrated that, after chronic ethanol feeding, selectively bred withdrawal-seizure-prone (WSP) mice displayed significantly higher levels of NMDA receptors in their brain tissue compared to withdrawal-seizure-resistant (WSR) mice, again relating susceptibility to withdrawal seizures to changes in, and/or inherent differences in, NMDA receptors.
We have used the information gathered from our studies to engineer novel medications for ethanol withdrawal which may overcome certain drawbacks of currently available treatments. We have designed and synthesized compounds which can simultaneously act as NMDA receptor antagonists and use-dependent blockers of voltage-sensitive sodium channels. We have shown these compounds to be effective as anticonvulsant agents and particularly effective in preventing ethanol-withdrawal induced seizures and convulsions. These compounds also have been shown to protect cells in culture from ethanol-withdrawal induced excitotoxicity.
Our early studies of brain adenylyl cyclase systems led us to conclude that adenylyl cyclase may have an important role in alcohol tolerance, as well as in the acute actions of ethanol. This conclusion resulted from our studies on lesioning particular noradrenergic pathways in brain of mice and blocking the development of alcohol tolerance to the incoordinating actions of ethanol. We then showed that we could reinstate the development of alcohol tolerance by activation of adenylyl cyclase systems in the brains of the lesioned animals.
Since adenylyl cyclase (AC) is not one, but a family of, enzymes, we more recently pursued cloning and characterizing novel forms of adenylyl cyclase and, in doing so, found a particularly ethanol-sensitive form of adenylyl cyclase, AC VII. When AC VII was expressed in cultured HEK 293 cells, the addition of intoxicating concentrations of ethanol could double the activity of AC VII in these cells (see Figure 1). We recently demonstrated that the ethanol-induced potentiation of this particular adenylyl cyclase activity may be regulated by PKC-mediated phosphorylation of this enzyme. We generated antibodies to AC VII to localize this protein in brain and found it to be highly localized to cerebellar Purkinje neurons, cortical neurons, and interneurons of the hippocampus. We also found AC VII to be expressed in other body tissues and gathered evidence that AC VII was the major form of adenylyl cylcase expressed in platelet progenitor cells. This allowed us to pursue studies of AC VII activity in platelets of human alcoholics and control subjects as a possible reflection of brain adenylyl cyclase activity. Our studies with humans showed that alcoholics have lower platelet adenylyl cyclase activity and that platelet adenylyl cyclase activity is a heritable trait. We recently localized the gene for AC VII to human chromosome 16 (see Figure 2), and have found that this gene has a polymorphism which will allow population and family studies at a genetic level.
Currently, our laboratories are acting as the coordinating center for a World Health Organization/International Society for Biomedical Research on Alcoholism sponsored study on state and trait markers in alcoholism. This study involves the recruitment of subjects in five clinical centers around the world, a thorough psychiatric/medical characterization of these subjects, and collection of biologic samples, including lymphocytes, for assessment of markers which can identify current alcohol abuse or predisposition to alcoholism. The genetic association portion of this study focuses on a number of candidate genes for alcoholism, including the polymorphisms in AC VII, as possible predisposing factors for development of alcoholism.