Essential Synergy between Ca 2 + and Guanine Nucleotides in Exocytotic Secretion from Permeabilized Rat Mast Cells

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 special attention to the role of nucleoside triphosphates in the secretory reaction of streptolysin-O-permeabilized mast cells. We show that Ca2÷-dependent exocytosis does not require the presence of phosphorylating nucleotides but that a ligand able to bind at GTP regulatory proteins is essential. Materials and Methods All the methods used in this work have been described in earlier publications (21, 25). Mast cells were isolated from rat peritoneal washings by centrifugation (300 g, 15 min) through a cushion of BSA (35% wt/wt). They were washed by centrifugation and suspended at '~0.2 x 106 ml -~ in a solution (pH 6.8) comprising 137 mM NaC1, 2.7 mM KCI, 20 mM Pipes, 5.6 mM glucose, and BSA (1 mg ml-I). In all experiments except those illustrated in Fig. 3, the mast cells were finally resuspended in a glucose-free buffer and treated with metabolic inhibitors (5 lttM antimycin A plus 6 mM 2-deoxyglucose) 2 rain before (Fig. t) or at the time of permeabilization (Figs. 2, 4, and 6) with streptolysin-O (0.4 IU ml-~). In such experiments reagents used were also made up in glucose-free buffer. In practice we have found that these treatments (i.e., 2-rain preincubation with metabolic inhibitors, or addition of inhibitors at the time of permeabilization) are both equally capable of reducing the background release of histamine (at pCa5) to low levels (25). In a typical secretion experiment, 1 vol of cells (30 p.1) was added to 3 or 4 vol of a solution containing streptolysin-O, nucleoside triphosphate (as the Mg 2+ salt) and calcium buffer (3 mM EGTA final). After incubation for 10 min at 37°C, the cellular reactions were quenched by addition of ice cold 0.15 M NaCI, buffered at pH 7 with 10 mM K phosphate and sedimented by centrifugation at 2,000 g for 5 min at 4°C (the secretion terminates in 3 min [25] so the use of 10 rain incubations ensures that corn© The Rockefeller University Press, 0021-9525/87/07/191/7 $2.00 The Journal of Cell Biology, Volume 105, July 1987 191-197 191 on A ril 3, 2017 D ow nladed fom Published July 1, 1987