| This thesis examined both the acute and the chronic regulation of ion channels in fully developed adult bullfrog sympathetic ganglia (BFSG) neurons by neurotransmitters acting via G-protein coupled receptors.; A variety of neurotransmitters excite neurons by suppressing a ubiquitous, voltage-dependent, non-inactivating K+ conductance called the M-conductance (gM), however the mechanism responsible remains unknown. The results presented in this thesis are consistent with the involvement of the lipid and inositol phosphate cycles in the effects of P2Y, muscarinic cholinergic, and luteinizing hormone releasing hormone (LHRH)-receptor agonists on gM. Suppression of gM by all three agonists involved phospholipase C (PLC), but not ‘downstream’ products of PLC, such as inositol trisphosphate (IP3), Ca2+, or protein kinase C (PKC). Impairment of re-synthesis of phosphatidylinositol 4,5, bisphosphate (PIP2) slowed or blocked recovery of agonistinduced gM suppression. Neutralizing PIP2-M-channel interactions inhibited steady state gM and protecting PIP2 from hydrolysis by PLC reduced LHRH-induced suppression. Intracellular application of PIP 2 slowed the run-down of KCNQ2/3 currents expressed in COS-1 or tsA-201 cells and PIP2 produced a small potentiation of native M-currents. These are the results which might be expected if agonist-induced activation of PLC and the concomitant depletion of PIP2 contributes to the excitatory action of neurotransmitters that suppress gM.; This thesis also exploited the simple organization of BFSG to test whether LHRH, the neurotransmitter for the late slow e.p.s.p, could also exert long-term neurotrophic control of ion channel expression. Removing all in vivo sources of ganglionic LHRH for 10 days by cutting preganglionic C-fibers reduced Ca2+ current density. Dissociated BFSG B-neurons exhibited an increased ICa density after 6–8 days in the presence of LHRH. This increase was not associated with alterations in activation or inactivation kinetics of the Ca2+ channel conductance, and reflected an increase in N-type Ca2+ channel current. The increase in ICa density induced by LHRH was blocked by a transcription inhibitor, and inhibitors of Ras isoprenylation, MAPK-kinase, protein kinase A, protein kinase C and phospholipase C. These results suggest that chronic activation of G-protein coupled receptors in a fully-differentiated, adult sympathetic neuron in vitro or in vivo exerts long term-control of ion channel expression. |
