Two G-proteins act in series to control stimulus-secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphodiesterase and exocytosis.

Abstract

Abstract. Provision of GTP (or other nucleotidescapable of acting as ligands for activation of G-proteins) together with Ca 2+ (at micromolar concentrations) is both necessary and sufficient to stimulate exocytotic secretion from mast cells permeabilized with streptolysin-O. GTP and its analogues, through their interactions with Gp, also activate polyphosphoinositide-phosphodiesterase (PPI-pde generating inositol 1,4,5-trisphosphate and diglyceride [DG]). We have used mast cells labeled with [3H]inositol to test whether the requirement for GTP in exocytosis is an expression of Gp activity through the generation of DG and consequent activation of protein kinase C, or whether GTP is required at a later stage in the stimulus secretion sequence. Neomycin (0.3 mM) inhibits activation of PPI-pde, but maximal secretion due to optimal concentrations of guanosine 5"O-(3-thiotriphosphate) (GTP-'t-S) can still be evoked in its presence. When ATP is also provided the concentration requirement for GTP-u in support of exocytosis is reduced. This sparing effect of ATP is nullified when the PPI-pde reaction is inhibited by neomycin. We argue that the sparing effect of ATP occurs as a result of enhancement of DG production and through its action as a phosphoryl donor in the reactions catalyzed by protein kinase C. W,r~ have shown that rat mast ceils permeabilized with streptolysin-O undergo exocytotic secretion when provided with micromolar concentrations of Ca 2+ together with GTP, xanthosine triphosphate, or inosine triphosphate (18,19). These nucleotides are capable of acting as ligands for activation of G-proteins (5, 33). The combination of Ca 2+ + G~P is both necessary and sufficient for secretion to occur. There is no absolute requirement for the presence of any other water soluble metabolite though ATP has a sparing effect on the concentration requirements for both the Ca 2+ and the GTP. Since polyphosphoinositidephosphodiesterase (PPI-pde), t the enzyme that generates the second messengers inositol 1,4,5-trisphosphate and diglyceride (DG), is subject to control by a GTP-binding protein, Gp (9, 24, 26, 31), this represents one possible site of action whereby the effect of guanine nucleotides might act to control secretion in the permeabilized cells. It is now well understood that the water soluble product of PPI-txie activation, inositol 1,4,5-trisphosphate, mobilizes Ca 2+ from intracellular stores. DG is the activator of protein kinase C (4, 27). However, the existence of Gp, and a defined role for GTP in receptor activation mechanisms does not preclude 1. Abbreviations used in this paper: DG, diglyceride; GTP-y-S, guanosine 5'-O-(3-thiotriphosphate); IP, inositol phosphate; pale, phosphodiesterase; PIP, phosphatidylinositol monophosphate; PPI, polyphosphoinositide. the possibility that GTP might have other roles in the complete sequence of events which leads to exocytotic release of secretory materials.In the experiments reported here we have used [3H]inositol-labeled mast cells permeabilized with streptolysin-O to fo...

GTPγS and phorbol ester act synergistically to stimulate both Ca2+‐independent secretion and phospholipase D activity in permeabilized human platelets

“…Similar studies with other permeabilized cells, particularly neutrophils [3], HL-60 cells [4] and RINmSF cells [5], in which Ca2'-independent secretion was observed, and with mast cells in which Ca2* was required [6,7], have led Gomperts and colleagues to propose that an unidentified G protein, designated G,, may mediate GTPySinduced exocytosis (reviewed in [S]). Since treatment of rabbit platelets with PMA enhances the ability of GTPyS to stimulate PLD in membrane preparations PMA, phorbol 12-myristate 13-acetate; PA, phosphatidic acid; PEt, phosphatidyl-ethanol; BAPTA, l,f-bis(oaminophenoxy)ethane-N,N,N,N'-tetraacetic acid; PKC, protein kinase C [9], we have suggested that PLD may be the effector of G, in platelets [l].…”

ARF1‐regulated phospholipase D in human neutrophils is enhanced by PMA and MgATP

“…We have previously shown that in the absence of MgATP, the polyphosphoinositide levels drop significantly and re-addition of MgATP at millimolar concentrations restores these levels [28]. The enhancement with MgATP when ARF-regulated PLD is examined in its natural environment may reflect the increased availability of PIP,.…”

Activation of exocytosis by cross-linking of the IgE receptor is dependent on ADP-ribosylation factor 1-regulated phospholipase D in RBL-2H3 mast cells: evidence that the mechanism of activation is via regulation of phosphatidylinositol 4,5-bisphosphate synthesis

“…Mast cells were obtained and purified exactly as described previously [5]. The protocol for cytosol depletion was identical to that used for RBL-2H3 cells described above.…”

“…Studies of exocytosis from cells of haematopoietic origin, including mast cells, have indicated an essential role for a Gprotein (G E ) in addition to increases in Ca# + [4,5]. Subsequent work has revealed several candidate G-proteins that are involved in exocytosis.…”