The role of G-proteins and second messengers in neuromodulation by extracellular ATP: The chromaffin cell as a model

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

RGS3 mediates a calcium-dependent termination of G protein signaling in sensory neurons

G proteins modulate synaptic transmission. Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of Gα subunits, and thus terminate G protein activation. Whether RGS proteins themselves are under cellular control is not well defined, particularly in native cells. In dorsal root ganglion neurons overexpressing RGS3, we find that G protein signaling is rapidly terminated (or “desensitized”) by calcium influx through voltage-gated channels. This rapid desensitization is most likely mediated by direct binding of calcium to RGS3, as deletion of an EF-hand domain in RGS3 abolishes both the desensitization (observed physiologically) and a calcium-RGS3 interaction (observed in a gel-shift assay). A naturally occurring variant of RGS3 that lacks the EF hand neither binds calcium nor produces rapid desensitization, giving rise instead to a slower calcium-dependent desensitization that is attenuated by a calmodulin antagonist. Thus, activity-evoked calcium entry in sensory neurons may provide differential control of G protein signaling, depending on the isoform of RGS3 expressed in the cells. In complex neural circuits subjected to abundant synaptic inhibition by G proteins (as occurs in dorsal spinal cord), rapid termination of inhibition by electrical activity by EF hand-containing RGS3 may ensure the faithful transmission of information from the most active sensory inputs

Agonist trafficking of G(i/o)-mediated α(2A)-adrenoceptor responses in HEL 92.1.7 cells

1. The ability of 19 agonists to elevate Ca(2+) and inhibit forskolin-induced cyclic AMP elevation through α(2A)-adrenoceptors in HEL 92.1.7 cells was investigated. Ligands of catecholamine-like- (five), imidazoline- (nine) and non-catecholamine-non-imidazoline-type (five) were included. 2. The relative maximum responses were similar in both assays. Five ligands were full or nearly full agonists, six produced 20 – 70% of the response to a full agonist and the remaining eight gave lower responses (<20%) so that their potencies were difficult to evaluate. 3. Marked differences in the potencies of the agonists with respect to the two measured responses were seen. The catecholamines were several times less potent in decreasing cyclic AMP than in increasing Ca(2+), whereas the other, both imidazoline and ox-/thiazoloazepine ligands, were several times more potent with respect to the former than the latter response. For instance, UK14,304 was more potent than adrenaline with respect to the cyclic AMP response but less potent than adrenaline with respect to the Ca(2+) response. 4. All the responses were sensitive to pertussis toxin-pretreatment. Also the possible role of PLA(2), β-adrenoceptors or ligand transport or metabolism as a source of error could be excluded. The results suggest that the active receptor states produced by catecholamines and the other agonists are markedly different and therefore have different abilities to activate different signalling pathways