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The role of G-proteins and second messengers in neuromodulation by extracellular ATP: The chromaffin cell as a model
Abstract
Upon activation of the acetylcholine receptor (muscarinic or nicotinic), chromaffin cells secrete catecholamine, nucleotides and neuropeptides. The secretory granules contain 150 mM ATP. Once this ATP is secreted to the extracellular environment it can regulate the cell by a feedback mechanism. Our laboratory has shown that ATP can enhance the catecholamine secretion evoked by the nicotinic receptor or high K+ and that this effect can be blocked by pretreatment with cholera toxin. Exposure of the cells to ATP prior to stimulation causes inhibition of catecholamine secretion. This inhibitory effect can be prevented by pretreatment with pertussis toxin. The goal of this thesis is to study the signalling pathways of the ATP-evoked effects. I have identified the bacterial toxin-sensitive G-proteins coupled to the ATP receptors and their effectors. By using isolated membranes or intact cells, we found that the ATP receptors were coupled to G$\sb{\rm s}$, G$\sb{\rm i}$ and G$\sb{\rm o}.$ The roles of calcium influx and its release from internal stores have been assessed. ATP can evoke calcium entry and release from internal stores. ATP$\gamma$S, an analogue which mimics the ATP enhancement of secretion, induced IP3 turnover. ATP$\gamma$S + DMPP evoked an increase in IP3 turnover that was greatly potentiated by pretreatment with cholera toxin. ADP, which inhibits secretion, inhibited IP3 turnover. This inhibition was prevented by pretreatment with pertussis toxin. The enhancement and inhibition of phospholipase C evoked by correlated with the effects on secretion. These effects on the activity of phospholipase C might reflect changes in calcium influx evoked by ATP. Part of the ATP-evoked effects on secretion can be explained by the effects of this nucleotide on chromaffin cell calcium currents. Upon addition of ATP, or ADP, which mimics the ATP inhibitory effects, 97% showed a decrease in their long-lasting calcium current. This inhibition of the calcium currents was prevented by pretreatment with pertussis toxin. In 20% of the cells tested, stimulation with ATP caused an increase in the inward current and an enhancement of the long-lasting currents. These currents desensitized to control current in about 30 seconds. Pretreatment with cholera toxin slowed down the rate of desensitization of both enhanced currents.
Subject Area
Biochemistry|Neurosciences|Cellular biology
Recommended Citation
Diverse-Pierluissi, Maria A, "The role of G-proteins and second messengers in neuromodulation by extracellular ATP: The chromaffin cell as a model" (1992). Doctoral Dissertations Available from Proquest. AAI9219425.
https://scholarworks.umass.edu/dissertations/AAI9219425