THE JOURNAL zyxwvutsrqp OF BIOLOGICAL zyxwvutsrqponm CHEMISTRY IC’ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Val. 266, No. 11. Issue of April 15. pp. 6859-6865, 1991 zy Printed in U. zy S. zy A. Mechanisms of Mastoparan-stimulated Surfactant Secretion from Isolated Pulmonary Alveolar Type 2 Cells* (Received for publication, August 2, 1990) Martin Joyce-Brady$$, Jeffrey B. Rubins$, Michail P. Panchenkol, John Bernardo$, Mark P. SteeleS, Lukas Kolm$, Elizabeth R. Simonsll , and Burton F. Dickey$[[ Massachusetts zyxwvutsrqp 021 18 and the BLaboratory of Molecular Endocrinology, Cardiology Research Center, From the $Pulmonary Center and the IIDepartment of Biochemistry, Boston Uniuersity School of Medicine, Boston, Moscow, Union of Souiet Socialist Republics Mastoparan, a tetradecapeptide component of wasp venom, is a potent activator of secretion in a variety of cell types, and has been shown to activate purified G-proteins reconstituted into phospholipid vesicles with a preferential activation of Gi over G. (Higashi- jima, T., Uzu, S., Nakajima, T., and Ross, E. R. (1988) zyxw J. zyxwvutsrqp Biol. Chem. 263, 6491-6494). To identify the bio- chemical activities of mastoparan in a cellular system, we characterized the effects of mastoparan on signal transduction pathways in rat pulmonary alveolar type 2 epithelial cells, which synthesize and secrete pulmo- nary surfactant. Mastoparan inhibited adenylylcyclase activity in a manner that was dose-dependent (IC5, = 30 PM), but sensitive to neither guanine nucleotide nor pertussis toxin (PT). Mastoparan induced a PT-sensi- tive iscrease in cellular inositol trisphosphate and a rapid rise in cytosolic calcium released from intracel- lular stores; the time to onset of the calcium rise, but neither the rate nor the amplitude of the rise, were PT- sensitive, Mastoparan also caused a dose- (EC50 = 16 PM) and time-dependent activation of arachidonic acid release that was completely insensitive to pretreatment with PT. Secretion of pulmonary surfactant was in- creased by mastoparan approximately 8-fold over con- stitutivelevelsat 1 h with an EC,, = 20 p ~ , and mastoparan-stimulated secretion was partially sensi- tive to PT at late time points and to inhibitors of arachidonic acid metabolism, but not to the protein kinase C inhibitor H7. These findings are consistent with the activation of Gi proteins in type 2 cells bymastoparan, although the lack of predicted triphosphoguanine nucleotide and PT sensitivityfor some activities indicates that masto- paran does not act in a manner strictly analogous to liganded receptors or that some activities are not me- diated by activation of Gi. While mastoparan is a potent secretagogue in several cell types, its secretory activity appears to have only a limited dependenceon the acti- vation of Gi proteins in type 2 cells. *This work was supported by an American Lung Association research grant and a biomedical research support grant from Boston University (to M. J. B.) and United States Public Health Service AwardsHL07864 (to J. B. R,), HL19717 (to J. B.), DK31056 and HL19717 (to E. R. S.), and HL05503 and HL43161 (to B. F. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. S To whom correspondence should he addressed: Pulmonary Cen- ter K603, 80 E. Concord St., Boston, MA 02118. The T2 cell’ of the pulmonary alveolus synthesizes and secretes surfactant into the alveolar space (1, 2). This lipo- glycoprotein complex functions to maintain alveolar stability by reducing alveolar surface tension forces at end-expiration (3,4). T2 cell surfactant secretion occurs by both constitutive and regulated mechanisms (reviewed in Refs. 5-7). The only well characterized signal transduction pathway that stimu- lates surfactant secretion is the adenylylcyclase-coupled @- adrenergic receptor system. T2 cell stimulation with @ago- nists (1, 8, g), the G, activator CT (lo), the adenylylcyclase activator forskolin (11, la), or directly with dibutyryl CAMP (8) results in activation of the PKA (12) and a 2-fold increase in surfactant secretion over the basal rate. The presence of additional signaling pathways leading to surfactant secretion from the T2 cell is suggested by the 4- fold increase in secretion induced by the PKC activator TPA (13, 14) or by a calcium ionophore (15-18). It .would, however, be advantageous to stimulate transmembrane signaling path- ways at a point proximal to the generation of soluble intra- cellularsecondmessengers,since the relative activation of different effector systems in a particular cell may lead to an integrated cytosolic signal that is characteristic for that cell. For example, PKC may be physiologically activated by di- acylglycerols generated directly through the activity of a PIP2- PLC with the accompanying generation of IPiI and a calcium transient (19), or by diacylglycerols generated through the sequential activitiesof a phospholipase D and a phosphatidic acid phosphatase without the generation of IP:I (20). Since ’ The abbreviations used are: T2 cell, pulmonary alveolar type 2 epithelial cell; G-protein, a member of the family of transductional guanine nucleotide-binding regulatory proteins; G,, a G-protein that mediates the stimulation of adenylylcyclase by liganded receptors and isasubstrateforcholeratoxin-catalyzedADP-ribosylation;G,, a member of a subfamily of 40- and 41-kDa G-proteins that are suh- strates for pertussis toxin-catalyzed ADP-ribosylation and modulate the activities of a variety of second messenger-generating enzymes and channels; G,,, a 39-kDa G-protein that is also a substrate for pertussis toxin-catalyzed ADP-rihosylation; GTPyS, guanosine 5’- 0-(3-thiotriphosphate); GDPbS, guanosine 5’-0-(2-thiodiphosphate); PT, pertussis toxin; CT, cholera toxin; PLC, phospholipase C; PLA,, phospholipase A2; PKC, protein kinases C, a family of calcium and phospholipid-dependent serine/threonine protein kinases; PKA. the CAMP-dependent protein kinase: PIPr, phosphoinositol bisphos- phate; IP;,, inositol trisphosphate; PC, phosphatidylcholine; DPPC, dipalmitoyl phosphatidylcholine; PMSF, phenylmethylsulfonyl fluo- ride; IBMX, 3-isobutyl-1-methylxanthine; EGTA, [ethylene- his(oxyethylenenitrilo)]tetraacetic acid;Hepes,4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid; DEC, diethylcarbamazine; NDGA, nordihydroguaiaretic acid; TPA, tetradecanoylphorholacetate; Indo- 1 and Indo-l/AM, the free acid and the acetoxymethyl esterof indo- 1, respectively; KRP, Krebs-Ringer’s phosphate; LDH, lactate dehy- drogenase; FBS, fetal bovine serum; MEM, minimal essential me- dium; BSS, balanced salt solution. 6859