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Abstract
When loaded alongside GTP-gamma-S into ATP-permeabilized cells, neomycin, at concentrations below 1 mM, inhibits GTP-gamma-S-induced histamine secretion and phosphatidic acid formation (Cockcroft, S., and B. D. Gomperts, 1985. Nature (Lond.). 314: 534-536; Aridor, M., L. M. Traub, and R. Sagi-Eisenberg. 1990. J. Cell Biol. 111:909-917). However, at higher concentrations internally applied neomycin induces histamine secretion in a process that is: (a) dose dependent; (b) dependent on the internal application of GTP; (c) independent of phosphoinositide breakdown; and (d) inhibited by pertussis toxin (PtX) treatment. These results indicate that neomycin can stimulate histamine secretion in a mechanism that bypasses phospholipase C (PLC) activation and yet involves a PtX-sensitive GTP-binding protein (G protein). Unlike its dual effects, when internally applied, neomycin induces histamine secretion from intact mast cells in a dose-dependent manner. Half-maximal and maximal effects are obtained at 0.5 and 1 mM neomycin, respectively. This process is rapid (approximately 30 s), is independent of external Ca2+, and is associated with phosphatidic acid formation, implying that neomycin can activate histamine secretion by a mechanism similar to that utilized by other basic secretagogues of mast cells. Neomycin stimulates fourfold the GTPase activity of cholate-solubilized rat brain membranes in a PtX-inhibitable manner. In addition neomycin, as well as the basic secretagogues of mast cells, compound 48/80, and mastoparan, significantly reduce (by approximately 80%) the ADP ribosylation of PtX substrates present in rat brain membranes. Taken together these data suggest that neomycin can stimulate secretion from mast cells by directly activating G proteins that play a role in stimulus-secretion coupling. When internally applied, neomycin presumably stimulates secretion by activating a G protein that is located downstream to PLC. This G protein serves as a substrate for PtX.
Highlights
H ISTAMINE secretion from rat peritoneal mast cells can be triggered by the introduction of nonhydrolyzable analogues of GTP, such as GTP-3,S, into cells permeabilized with ATP (Gomperts, 1983)
Neomycin is known to bind strongly and selectively to inositol phospholipids (Schacht, 1978), thereby leading to inhibition of their metabolism. It appears that the persistent activation of a G protein that is coupled to a phosphoinositide-hydrolyzing phospholipase C (PLC) (Gp) brings about histamine secretion
We proposed that basic secretagogues of mast cells may directly activate a G protein that is located downstream from PLC (Gs), thereby inducing secretion in a mechanism that bypasses PLC activation
Summary
PA, phosphatidic acid; PLC, phospholipase C; PtX, pertussis toxin. and secreting histamine in the presence of neomycin (Howell et al, 1987). We have demonstrated (Aridor et al, 1990) that the basic secretagogues, compound 48/80 and mastoparan, are capable of stimulating secretion from ATP-permeabilized ceils in a process that is not accompanied by PA formation but is dependent on the internal application of GTP. We could further demonstrate that compound 48/80, like mastoparan (Higashijima et al, 1988), is capable of directly stimulating the GTPase activity of G proteins in a cell-free system. Based on these results, we proposed that basic secretagogues of mast cells may directly activate a G protein that is located downstream from PLC (Gs), thereby inducing secretion in a mechanism that bypasses PLC activation. We show that neomycin acts as a potent secretagogue of mast cells by directly activating G proteins involved in exocytosis
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