Compartmentation of muscarinic cholinergic receptors coupled to phosphoinositide hydrolysis.

This thesis describes an investigation of the relationship between stimulated phosphoinositide (PPI) hydrolysis and muscarinic cholinergic receptor (mAChR) regulation in human SK-N-SH neuroblastoma cells. These cells possess an abundance of mAChRs (m$\sb3$) exclusively coupled to PPI hydrolysis. Three temporally distinct responses were elicited upon mAChR activation: (1) a rapid (sec) and sustained release of inositol phosphates (IPs); (2) a reversible sequestration (t$\sb{1/2}$ $\sim$ 7 min) of mAChRs from the cell surface to a more lipophilic cellular compartment; and (3) a down-regulation of mAChRs (t$\sb{1/2}$ $\sim$ 4 h) accompanied by a loss of stimulated PPI hydrolysis. Functional coupling of sequestered mAChRs to phospholipase C (PLC) was assessed in intact cells at 10$\sp\circ$C. At this temperature IP production was measurable, but mAChR cycling between sequestered and cell surface domains was blocked. Prior sequestration of mAChRs resulted in a comparable reduction of stimulated PPI hydrolysis when assayed with either lipophilic or hydrophilic agonists. Upon rewarming cells to 37$\sp\circ$C, sequestered mAChRs returned to the surface, and full PPI hydrolysis was regained. These observations suggest that sequestered mAChRs in SK-N-SH cells are incapable of activating PLC. In the final part of this thesis, the role of PPI hydrolysis in mAChR sequestration was investigated either by depletion of extracellular Ca$\sp{2+}$ or following incubation of the cells with U-73122, an aminosteroid inhibitor of PLC-mediated events. Whereas a reduction of stimulated IP production upon removal of Ca$\sp{2+}$ did not affect mAChR sequestration, the addition of U-73122 inhibited both events to a similar extent. To further probe its site of action, U-73122 was introduced into digitonin-permeabilized cells. The aminosteroid inhibited agonist- and GTP$\gamma$S-stimulated PPI hydrolysis but not direct Ca$\sp{2+}$ stimulation of PLC; moreover, this agent was not effective in modulating adenylate cyclase activity. These results suggest a site of action for U-73122 at the putative G protein that regulates PLC. It is proposed that, whereas IP production is not a prerequisite for mAChR sequestration, interaction of the mAChR with G$\sb{\rm p}$ is an obligatory step.PhDPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105796/1/9208668.pdfDescription of 9208668.pdf : Restricted to UM users only