Abstract. Rat mast cells, pretreated with metabolic inhibitors and permeabilized by streptolysin-O, secrete histamine when provided with Ca 2+ (buffered in the micromolar range) and nucleoside triphosphates. We have surveyed the ability of various exogenous nucleotides to support or inhibit secretion. The preferred rank order in support of secretion is ITP > XTP > GTP >> ATE Pyrimidine nucleotides (UTP and CTP) are without effect. Nucleoside diphosphates included alongside Ca 2+ plus ITP inhibit secretion in the order 2'-deoxyGDP > GDP > o-GDP > ADP ---2'deoxyADP ----IDP.Secretion from the metabolically inhibited and permeabilized cells can also be induced by stable analogues of GTP (GTP-T-S > GppNHp > GppCH2p) which synergize with Ca 2+ to trigger secretion in the absence of phosphorylating nucleotides. ATP enhances the effective affinity for Ca 2+ and GTP analogues in the exocytotic process but does not alter the maximum extent of secretion. The results suggest that the presence of Ca 2+ combined with activation of events controlled by a GTP regulatory protein provide a sufficient stimulus to exocytotic secretion from mast cells.T o learn about the train of postreceptor processes which control exocytosis in secretory cells, or indeed, almost any other form of cellular activation, it is advantageous to gain access, by permeabilization of the plasma membrane, to the intracellular environment. A number of procedures for membrane permeabilization have been devised that allow secretory cells to respond to extracellular ligands and intraceUular second messengers (such as Ca 2÷, GTP, etc.) applied alone and in combination (20,32). From such studies, two separate pictures have emerged. In platelets (23, 31), the Ca 2÷ requirement for exocytosis is shifted to lower concentrations when GTP analogues are present. Since phorbol esters and thrombin (24) modulate Ca2+-induced secretion in a similar manner, it has been concluded that the effect of internalized GTP (analogues), which activate polyphosphoinositide phosphodiesterase (13), is exerted through diacylglycerol, and hence phosphorylation by protein kinase C. In other systems such as rabbit neutrophils (5), it appears that Ca 2+ and GTP analogues are both independently capable of activating exocytotic secretion by a mechanism not involving activation of protein kinase C.We recently reported that mast cells permeabilized by treatment with streptolysin-O (a streptococcal cytotoxin) undergo an exocytotic secretory reaction, releasing histamine and I~-N-acetylglucosaminidase in response to Ca 2÷ buffered at concentrations in the micromolar range (25). The lesions generated by streptolysin-O in liposomal membranes have a diameter in excess of 12 nm and are sufficient to permit fluxes of large proteins such as catalase, urease, and ferritin (12). Mast cells permeabilized with streptolysin-O lose >65 % of lactate dehydrogenase within 5 min yet remain responsive to step changes of Ca 2+, from pCa8 to pCa5 up to 10 min after permeabilization (25). In the present paper we pay s...
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...
The secretory process is a coordinated cellular response, initiated by occupation of surface receptors and comprising an ordered sequence of biochemical steps subject to multiple controls. Conceptually we can divide the sequence into two main sections comprising early, receptor-mediated events leading to generation of intracellular second messengers, and later events leading to membrane fusion and exocytosis. With the discovery that occupation of Ca2+ mobilising receptors leads to activation of polyphosphoinositide phosphodiesterase (PPI-pde) through the mediation of a G-protein (Gp), all the early events can be ascribed to the plasma membrane. Investigation of the exocytotic stage of secretion has been simplified by the use of permeabilised cells in which the composition of the cytosol can be precisely controlled. We have used streptolysin-O, a bacterial cytolysin which generates protein-sized pores in the plasma membrane, to investigate the exocytotic mechanism of rat mast cells. We find that in addition to the activation of PPI-dpe, GTP also acts in concert with Ca2+ at, or close to, the exocytotic site. Exocytosis can occur after substantial depletion of cytosol lactate dehydrogenase and 3-phosphoglycerate kinase indicating that soluble cytosol proteins are unlikely to play any role. There is no absolute requirement for ATP or phosphorylating nucleotide in exocytosis though when present the effective affinities of the two obligatory effectors (i.e. Ca2+ and GTP) are substantially enhanced.
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