Biochem. J. (1988) 250, 375-382 (Printed in Great Britain 375 Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin 0 Jane STUTCHFIELD and Shamshad COCKCROFT Department of Experimental Pathology, School of Medicine, University College London, University Street, London WCIE 6JJ, U.K. The non-differentiated HL60 cell can be stimulated to secrete when Ca2" and guanosine 5'-[y-thio]- triphosphate (GTPyS) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentration- dependence on Ca2" between pCa 8 and pCa 5. The half-maximal requirements for Ca2" for PPI-pde activation and secretion are pCa 6.4 + 0.1 and pCa 6.2 + 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTPyS > guanosine 5'-[/y-imido]- triphosphate > guanosine 5'-[y-methylene]triphosphate > XTP - ITP, but the maximal response achieved by each compound compared with GTPyS is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTPyS-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTPyS in stimulating secretion, suggesting that the effect of GTPyS cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2" and GTPyS requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (Gp) and the other for activating exocytosis (GE), acting in series. INTRODUCTION already proposed as the effectors for initiating secretion Guanine nucelotide regulatory proteins are inter- [15,16]. In neutrophils [11], adrenal chromaffin cells (12] mediary molecules that transduce information between and RINm5F cells [13], it has been shown that GTPyS incoming signals (e.g. hormone receptor) and target can stimulate Ca2l-independent secretion, conditions proteins (such as adenylate cyclase) [1,2]. One such which would not stimulate PPI-pde [11,12,17]. In mast system involves the activation of polyphosphoinositide cells, secretion triggered by Ca2" plus GTPyS is resistant phosphodiesterase (PPI-pde) which is responsible for to inhibition by neomycin [14], an inhibitor of PPI-pde. hydrolysing PIP2 (phosphatidylinositol bisphosphate) We have designated this putative G-protein GE [11]. and possibly PIP (phosphatidylinositol phosphate) [3,4]. To differentiate further the effects of guanine nucleo- The products of hydrolysis, IP3 (inositol 1,4,5-tris- tides between PPI-pde activation and exocytosis, we have phosphate) and DG (diacylglycerol), are intracellular used the clonal line, HL60 cells. These cells are a human signals involved in mobilizing cytosolic Ca2" and activat- promyelocytic cell line which can be terminally differen- ing protein kinase C respectively [5,6]. PPI-pde activation tiated either into a neutrophil-like or a macrophage-like is coupled to receptors by a putative G protein, Gp [3,4]. cell depending on the inducer [18-20]. We demonstrate Evidence for this includes activation of PPI-pde by stable here that the undifferentiated cell has the intracellular GTP analogues and their ability to potentiate the effects machinery to undergo exocytosis and PPI-pde activation of receptor-directed agonists in plasma membranes or although cell surface receptors for agonists such as fMet- permeabilized cell preparations [3,4,7-101. Leu-Phe are absent. GTP and its stable analogues and A role for guanine nucleotides other than their role in Ca2" buffers were introduced directly into the cytosol by activating PPI-pde has been described recently in the permeabilizing the plasma membrane with streptolysin process of exocytosis in a variety of permeabilized cells 0, a bacterial cytolysin [14,21,22]. We are able to [11-14]. Exocytosis ofsecretory granules is one important demonstrate a dual role for guanine nucleotides, one for example of cell activation where Ca2+ and DG are PPI-pde activation and the other involved in exocytosis. Abbreviations used: PPI-pde, polyphosphoinositide phosphodiesterase; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol bisphosphate; IP1, inositol monophosphate; IP2, inositol bisphosphate; IP3, inositol trisphosphate; GTPyS, guanosine 5'-[y-thiojtriphosphate; GppNHp, guanosine 5'-[fly-imido]triphosphate; GppCH2p, guanosine 5'-[fly-methylene]triphosphate; G protein, guanine nucleotide regulatory protein; DG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate. Vol. 250 376 J. Stutchfield and S. Cockcroft METHODS AND MATERIALS to Medium 199 for the last 18-24 h, which varied from 24 to 36% [mean 27 % ± 7 (S.D.), n = 20]. In later Materials experiments we supplemented the Medium 199 with Streptolysin 0 was obtained from Wellcome Diag- bovine serum albumin (3.5 mg/ml) (as a substitute for nostics (Dartford, Kent, U.K.). RPMI-1640, Medium the foetal calf serum) and we found that secretion was no 199, glutamine, penicillin and streptomycin were all longer impaired. The formation of inositol phosphates obtained from Flow Laboratories. Foetal calf serum was was unaffected by the change in protocol. obtained from Imperial Laboratories. [3H]Inositol was For the time-course experiments, 100,1 aliquots were obtained from either NEN or Amersham. All nucleotides removed at the indicated times into 1 ml of ice-cold were obtained from Boehringer Mannheim except XTP 0.15 M-NaCl buffered at pH 7 with 10 mM-potassium and dGDP which were obtained from Sigma. phosphate and the cells processed as indicated above. Methods Calcium buffers. Ca2l was buffered with 3 mM-EGTA Culturing of HL60 cells. HL60 cells were grown in at concentrations between pCa 8 and pCa 5 and free RPMI-1640 supplemented with 15 % heat-inactivated Mg2+ was maintained at 2 mm. CaEGTA buffers were foetal calf serum, L-glutamine (2 mM), streptomycin prepared as described previously [25]. For nominally (50,ug/ml) and penicillin (50 i.u./ml). The cells were zero Ca2+, 3 mM-EGTA was used. The maximum error passaged at starting densities of (0.2-0.3) x 106 cells/ml due to varying the concentration of ATP in the range and maintained in suspension culture in an air/CO2 0-10 mm was <0.02 pCa. (19: 1) humidified atmosphere at 37 'C. The cell cultures were diluted every 2-3 days so that the cell density was Inositol phosphate determinations. Inositol phosphate maintained at (1-2) x 106 cells/ml. formation was used as an indicator ofPPI-pde activation. Either a 100,u aliquot of the supernatant was loaded Labelling of HL60 cells with I3Hlinositol. HL60 cells directly onto Dowex columns, or the cells plus the were harvested by centrifugation and resuspended at supernatant were quenched with chloroform/methanol a density of 106 cells/ml in 50 ml of Medium 199 and the aqueous top phase used for the analysis of supplemented with glutamine (2 mM), streptomycin inositol phosphates by ion-exchange chromatography on (50 #sg/ml), penicillin (50 i.u./ml), transferrin (5 ,ug/ml), Dowex columns as described previously [14,26]. Both insulin (5,ug/ml) and [3H]inositol (1,Ci/ml) and procedures gave essentially the same results. All three incubated as above for 18-24 h. Medium 199 was chosen inositol phosphates were routinely collected and since as the labelling medium because of its low levels of the major increase was always in 'P3 (see Fig. 3), data for inositol. For the same reason foetal calf serum was ex- IP3 are only presented for clarity in the remainder of the cluded and replaced with the growth factors insulin and experiments. The changes in lP2 and IP1 were similar to transferrin. the changes in 'P3 for all the experiments. In those experiments where nucleotides were used at Cell permeabilization and stimulation. For the study of concentrations up to 10 mm (see Figs. 3 and 7), we secretion, cells grown in RPMI were used. Labelled cells checked the possibility that such high concentrations of were used when activation ofPPI-pde was simultaneously nucleotides may affect the binding of inositol phosphates monitored. For a typical experiment, the cells were to the Dowex columns. The presence of 10 mM-XTP, washed three times in a buffered salt solution (pH 6.8) -ATP or -ITP (added to samples subsequently) did not which contained 137 mM-NaCl, 2.7 mM-KCl, 20 mm- affect the binding of inositol phosphates on the Dowex Pipes, 5.6 mM-glucose, 1 mg of albumin/ml and 15 SM- columns. EGTA. LiCl (10 mM) was added if inositol phosphate formation was also monitored. Cytochalasin B (2 ,ug/ml Expression of data. All determinations were carried final) was added to the cells 2 min before transferring out in duplicate except for the time-course experiments, 50 ,u ofcells to 4 vol. ofa mixture containing streptolysin and experiments were repeated on at least three occasions. O (0.4 i.u./ml final, except for Fig. 1), CaEGTA buffers (3 mM EGTA final) and nucleotides as detailed in the text and Figure legends. In those experiments where the cells were metabolically inhibited, glucose was omitted from RESULTS the buffer and the cells were pretreated with metabolic Fig. 1(a) illustrates the concentration-dependence of inhibitors (5 ,uM-antimycin A plus 6 mM-2-deoxyglucose) streptolysin 0 for lactate dehydrogenase release and ,- for 4 min. In such experiments reagents used were also glucuronidase secretion in the presence of pCa 5 only or made up in glucose-free buffer.