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J. Biol. Chem., Vol. 266, Issue 35, 23856-23862, 12, 1991
AK Thompson, SP Mostafapour, LC Denlinger, JE Bleasdale and SK Fisher
The relationship between muscarinic receptor activation of phosphoinositide
hydrolysis and the sequestration of cell surface muscarinic receptors has
been examined for both intact and digitonin- permeabilized human SK-N-SH
neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17
beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]
hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the
inhibition of oxotremorine-M-stimulated inositol phosphate release and of
Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was
directly activated by the addition of the calcium ionophore ionomycin,
inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to
intact cells also inhibited the agonist-induced sequestration of cell
surface muscarinic receptors and their subsequent down-regulation with an
IC50 value (4.1 microM) similar to that observed for inhibition of inositol
phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated
phosphoinositide hydrolysis was inhibited by depletion of extracellular
Ca2+, no reduction in the extent of receptor sequestration was observed.
When introduced into digitonin-permeabilized cells, U-73122 more markedly
inhibited inositol phosphate release elicited by either oxotremorine-M or
guanosine-5'-O- (3-thiotriphosphate) than that induced by added Ca2+.
Addition of oxotremorine-M to permeabilized cells resulted in muscarinic
receptor sequestration and down-regulation. Both the loss of muscarinic
acetylcholine receptors and activation of phosphoinositide hydrolysis in
permeabilized cells were inhibited by the inclusion of guanosine-5'-
O-(2-thiodiphosphate). The results indicate that the agonist-induced
sequestration of muscarinic acetylcholine receptor in SK-N-SH cells
requires the involvement of a GTP-binding protein but not the production of
phosphoinositide-derived second messenger molecules.
The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation
Neuroscience Laboratory, University of Michigan, Ann Arbor 48104-1687.
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