Biochemical zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Pharmacology. Vol. 42, No. 6. pp. 1267-1271.1991 Printed in Great Britain. cmi-2952/91 $3.00 + 0.00 @ 1991. PergamonPress plc PERTUSSIS AND CHOLERA TOXINS INHIBIT PROSTAGLANDIN SYNTHESIS IN RAT ASTROCYTE CULTURES AT DISTINCT METABOLIC STEPS PETER J. GEBICKE-HAERTER,* ANGELIKA SCHOBERT and zyxwvutsrqponmlkjihgfedcbaZYXWV GEORG HERTTING Institute of Pharmacology, University of Freiburg, Hermann-Herder-Str. 5, D-7800 Freiburg i.Br., Germany (Received 26 December 1990; accepted 19 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR M ay 1991) zyxwvutsrqponmlkjihgfedcbaZYXW Abstract-The influence of pertussis and cholera toxin-sensitive G-proteins in the prostaglandin synthetic pathway has been investigated. Prostaglandin D2 (PGD2) synthesis was stimulated by the calcium ionophore A23187, the phorbol ester tetradecanoylphorbol acetate (TPA), or by extracellular ATP. Pretreatment of cultures with pertussis toxin (Ptx) resulted in a partial inhibition of PGDl synthesis in both stimulated and unstimulated cells. A23187-stimulated PGDl synthesis was affected less than ATP- or TF’A-stimulated synthesis. Furthermore, Ptx also inhibited A23187-, ATP-, and TPA-stimulated arachidonic acid release. Basal and stimulated PGD, synthesis were also inhibited, when cultures were preincubated with cholera toxin (Ctx). Here, ATP-stimulated synthesis was affected the most. Arachidonic acid release, in contrast, was enhanced by cholera toxin, even without addition of stimuli. These data suggest that regulation of prostaglandin synthesis in rat astrocyte cultures involves Ptx- and Ctx-sensitive G-proteins. Ptx substrates affect events at or proximal to phospholipase AZ, whereas Ctx substrates influence events proximal or distal to phospholipase Al. A very early step in intracellular signaling pathways entails an activation of receptor-coupled GTP- binding proteins. Activated G-proteins are able to either up- or down-regulate the activities of cellular key enzymes, or regulate ion channels [l-3]. Pertussis and cholera toxins (Ptx and Ctxt) have proved to be an invaluable aid in elucidating the involvement of G-proteins in intracellular signaling cascades by their specific ability to ADP-ribosylate the a-subunits of a variety of G-proteins, resulting in their irreversible inactivation or long-lasting activation, respectively [ 1,4]. Pertussis toxin sensitive G- proteins have not only been implicated as inhibitors of adenylate cyclase (GJ but also as regulators of phospholipase C [5-81 and phospholipase A2 activities [ 1,9, lo]. The present report gives evidence of an involvement of both Ptx- and Ctx-sensitive G- proteins in the regulation of prostaglandin synthesis in astroglial cultures. Pertussis toxin substrate(s), however, appears to control some early event in the biosynthesis of prostaglandins, whereas Ctx substrate(s) may interfere primarily with later events. MATERIALS AND METHODS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF M aterials. Ptx and Ctx were purchased from List laboratories (Campbell, CA, U.S.A.); [3H]PGD2 and [3H]arachidonic acid were from Amersham and * To whom correspondence should be addressed. t Abbreviations: AA, arachidonic acid; BSA, bovine serum albumin; DAG, diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; DPBS, Dulbecco’s phosphate buffered saline; IP,, inositol trisphosphate; PGD*, prostaglandin D2; LPS, lipopolysaccharide; PIP,, phos- phoinositoldiphosphate; PLA,, phospholipase Al; F’tx, pertussis toxin; Ctx, cholera toxin; TPA, tetradecanoyl phorbol acetate. Buchler, (Braunschweig, Germany); fatty acid free BSA, ATP, TPA and A23187 were from the Sigma Chemical Co. (Deisenhofen, Germany); LPS (Salmonellu ty phii) and fetal calf serum were from Sebak/Pan Systems (Aidenbach, Germany); and DMEM was from Gibco (Eggenstein, Germany). PGD2 antisera were made according to a procedure described previously [ 111. Cross-reactivity with PGE2 and other prostaglandins, or with AA was less than 1%. Cell cultures. Astroglial cultures were prepared from newborn rats as described elsewhere [12] and maintained at precisely 10 ng LPS/mL media [13]. Prostaglandin release and radioimmunoassays. Cultures were preincubated in HEPES-buffered DMEM, pH7.4 and cells were then stimulated for another 15 min in the same media containing substances as indicated. Reactions were stopped by mixing supernatants into ice-cold phosphate-buffered gelatin solution, pH7.4. Cells were solubilized in 0.1 M NaOH and protein was determined according to Lowry et al. [14]. Supematant-gelatin (200- 500 pL, 0.1%) mixture was used for radio- immunoassays specific for PGD,, as described previously [ll]. Arachidonic acid release. Cells were incubated overnight in [3H]AA (0.25 pCi/mL; sp. act. 207 Ci/ mmol) and 10 ng/mL unlabeled AA (maximum 3H- incorporation was achieved at this concentration, which probably ensured an even distribution in all cellular compartments). After removal of the media, they were incubated sequentially in DPBS (5 min), DPBS/l% BSA (fatty acid-free) (5 min) and stimulated with ATP, A23187, or TPA in PBS/ BSA (30min). Supematants were transferred into scintillation vials and radiowtivity determined. AA release was calculated as the percentage of total 1267