The purpose of this project is to determine second messenger pathways involved in receptor regulation of neurotransmitter levels. Using bovine adrenal medullary cells, Dr.
R aim o Tuominen in our lab had found that angiotensin II (AII) and nicotine increased protein kinase C (PKC) translocation and diacylglycerol (DAG) levels in the cells. As this is the only second messenger pathway which our lab has shown to be increased long-term, this project was continued and expanded. Following C 3 H]choline and [3 H]inositol incorporation and breakdown in these cells, it appears that phosphatidylcholine is the likely source of long-term (10 minutes or later) DAG formation. Using [3H]phorbol libutyrate binding to follow PKC translocation, agonists which stimulate Ca2+ influx rather than intracellular Ca2+ mobilization were found to be more efficacious in stimu- lating [3 H]PdBu binding in BAM cells. While Ca2+ mobilizing pathways are distinct for different agonists, it is unclear whether the DAGs formed and the PKCs translocated are the same for each agent. AII stimulates C 3 H]PdBu binding through an extracellular Ca2+- and omegaCgtx-sensitive pathway, but the effect is small and transient in contrast to the effect of nicotine and depolarizing stimuli. The same picture emerges with [3H]PdBu binding to C6 glioma cells, SMS-KCNR neuroblastoma cells and primary cultures of hippocampal and striatal cells (other cells in which enkephalin MRNA levels may be regulated by Ca2+ and/or PKC): extracellular Ca2+ appears much more important for PKC translocation than is intracellular Ca2+ mobilization. An interesting finding resulting from this technique was that dopamine increased [3 H]PdBu binding in striatal cells through a novel D-1 receptor, which was almost as effective as well-characterized Ca2+-Mobilizing agonists such as norepinephrine and excitatory amino acids. The dopamine response was antagonized by delta and mu (but not kappa) opiate receptor agonists; it is unclear whether this antagonism is specific to PKC activation and [3H]PdBu binding.
