European Journat of Pharmacology - MolecularPharmacology Section, 206 (1991) 309-314 309 © t991 Elsevier Science Pubhshers B.V. 0922~4106/91/S03.50 ADONIS 09224106910009d2 EJPMOL 90163 Effects of mastoparan and related peptides on phosphoinositide breakdos,~nain I~-60 cells and ceil-free preparations Fabian Gusovsky, David G. Soergel and John W. Daly Laboratory of Bioorganic Chemistry. NIDDK. Nanonal lmt~tutes of Health, Bethesda. MD 20892, U.S.A. Received21 December 1990.accepted 2 ga-nuaD" 199t In differentiated HL-60 cells the amphiphilic peptide mastoparan indut.as a do.-dependent stimulation of phosph~inositide breakdown with an ECs0 value of 9 pM. Such stimulation can be markedly reduced by pretreatment of the cells with pertussis my, in (100 ng/ml, 2 h). In membranes obtained from differentiated HL-60 cells, guanine nucleotides stimulate the formation of IP2 and IP3. Calcium ions also induce phosphoinositide breakdown in this preparation independent of the presence of guanine nucleotid~. In HL-60 cell membranes, mastoparan inhibited GTPl, S-stimulation of phosphoinositide breakdown v, rith an IC50 value of 3/tM, Such inhibitory activity of mastoparan also was present in membranes from cells pretreated with pertussks toxin. CMcinm-induced stimulation of phosphoinositide breakdown was not significantly inhibited by mastoparan. The analogs mastopa~an-X and polistes mastoparan had similat inhibitory activity, whereas the analog des-lteLAsn2-mastoparan was inactive. In permeabilized HL~60 cells mastoparan also inhibited phosphoinosifide breakdown. Another amphipbAlic peptide, mNittin~ was inactive in HL-60 intact cells, but similar to mastoparan, inhibited guanine nucleotide-induced phosphoinosifide breakdown i:a HL.~.~ cetJ membranes and permeabilized cells. Thus, mastoparan peptides can stimulate phosphoinositide breakdown in intact HL-60 ceils, probably through the interaction with a guanine nucleofide binding protein. In permeabifized cells and in ceil merabranes, mastoparan induces inhibition of guanine nucleotide-mediatexJ phosphoinosltide breakdown presumably through an interaction with an intracetinlar site. The inhibitory action of mastoparan and melittin is probably related te the amphiphific character of these pepfide~. MastoparaD; Phospholipase C; G proteins; Amphiphilic peptides 1, htrodudio: Mastoparan, an amphiphilic peptide isolated from wasp venom, is a potent secretagogue in mast cells and other cell types (Higashijima et al., 1987; Nakajima et a!., 1986). Mastoparan stimulates phosphoinositide breakdown in mast cells (Okano et at., i985) and cer- tain other cells (Choi and Daly, 1990; Wojcikiewicz and Nahorski, 1989), suggesting that, like many other secretagogues, stimulation of phosphoinositide break- down by mastoparan and a resultant release of calcium from intracellular sites by IP3, may underlie the effects of mastoparan on release processes. The effects of mastoparan on phosphoinositide breakdown now were examined in differentiated HL-60 cells. In intact HL-60 cells, mas~oparan and related peptides stimulated phospholipase C activity. However, in permeabilized HL-60 cells and membranes. mastoparan, its analogs mastoparan X and polistes Correspondence to: Fabian Gusovsky, LBC. NIDDK, 8tdg 8, Room 1A-15, NIH, Bethesda~MD 20892, U.S.A. mastoparan and the amphiphilic peictide melittin, in- hibited guanine nucleotide-stimulated phospho!ipase C activity. 2, Materials and methods Z 1. Cell culture HL-6O cells were ~own in suspeesion in RPMI 1640 medium containing ! 0% fetal calf serum and antibiotics. Cells were differentiated and labelled with [~H]inosiml in inositol-free gro~ing medium containing 100 aM dibutyryl cyclic AMP and 10 pzCi/ml for 48 h before experiments. Z2. Phosphoinositide breakdown in HL-60 cells The procedure was essentially as described by Brandt et ~. (1985). Labelled HL-60 cells were washed with buffer A (118 mM NaCI, 4.7 mM KCL 3 mM CaCI~, 1.2 mM MgSO~, L2 mM KH2PO 4, 0.5 mM EDTA, 10 ram glucose and 20 raM HEPES~ pH 7.4) containing 10