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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 7320-7324, October 1986 Cell Islet-activating protein inhibits - and - but not bradykinin- or calcium ionophore-induced synthesis in bovine endothelial cells (pertussis toxin//ADP-ribosylation/calcium ionophore A23187) MIKE A. CLARK, THERESA M. CONWAY, C. FRANK BENNETT, STANLEY T. CROOKE, AND JEFFREY M. STADEL Department of Molecular Pharmacology, Smith Kline and French Laboratories, P.O. Box 7929, Philadelphia, PA 19101 Communicated by James M. Sprague, July 1, 1986

ABSTRACT Incubation of the bovine endothelial cell line, Increases in synthesis in response to LTC4 (2) and CPAE, with leukotriene D4, leukotriene C4, bradykinin, or the LTD4 (7) appear to be mediated in that these effects calcium ionophore A23187 results in the release of arachidonic can be inhibited by specific antagonists. Studies in several acid metabolites including 6-keto- Fla, the stable laboratories have directly identified membrane-localized recep- metabolite of prostacyclin. Pretreatment of these cells with the tors for the peptidyl leukotrienes LTC4 and LTD4 (8-16). These pertussis toxin islet-activating protein (IAP) results in a dose- receptors have been found in guinea pig and rat (8-12), dependent inhibition of the release of arachidonic acid metab- guinea pig heart (13, 14), and human lung (15, 16). Furthermore, olites and prostacyclin in response to leukotriene D4 and in membranes isolated from smooth muscle cell lines, LTC4 leukotriene C4. In contrast, similar responses evoked by binding sites have clearly been identified (2, 17). Although more bradykinin or ionophore were not significantly altered by the difficult to characterize, LTD4 receptors have also been shown IAP pretreatment of the cells. IAP in the presence of [32P]NAD to coexist in these cell lines (2). In membranes isolated from specifically [32P]ADP-ribosylates a 41-kDa protein in mem- guinea pig and human lung, LTD4 receptors have been shown branes prepared from CPAE cells. Pretreatment of the intact to be modulated by sodium ions as well as guanine nucleotides cells with UAP resulted in a dose-dependent inhibition of (8, 18). LTD4 receptors solubilized from guinea pig lung retain subsequent 32p labeling ofthe toxin substrate in the membranes their ability to be modulated by sodium ions and guanine and correlates with the uncoupling of the leukotriene respons- nucleotides (19). These studies suggest that the membrane es. These results suggest that the 41-kDa UAP substrate, localized receptors for LTD4 may be coupled via guanine presumably a guanine nucleotide regulatory protein, mediates nucleotide binding proteins. the response of CPAE cells to leukotriene D4 and leukotriene Islet-activating protein (IAP), a toxin isolatedfromBordetella C4, but not to bradykinin or the calcium ionophore. pertussis, inhibits the biological effects of certain agonists that stimulate the release of arachidonic acid from phospholipids Leukotriene C4 (LTC4) and leukotriene D4 (LTD4) induce (20-22). The only known mechanism of action of IAP is the contraction in several smooth muscle cell lines in culture (1, specific ADP-ribosylation ofthe 41-kDa subunit ofthe guanine 2). We have reported that the interaction of LTC4 and LTD4 nucleotide regulatory protein (NJ) that mediates hormonal with their receptors induces contraction via a process that inhibition of adenylate cyclase (23-28). The effect of the toxin requires RNA and protein synthesis (2). The protein(s) is to attenuate Ni-mediated inhibition of the cyclase . synthesized appear to be involved in activating phospholip- The observation that IAP can also modulate agonist promoted ase A2 resulting in the liberation of arachidonic acid from arachidonic acid release and that this effect correlates with the phosphotidyl choline (3). In cell lines that contract when modification of a 41-kDa membrane protein (20-22) suggests a challenged with peptidyl leukotrienes, the liberated arachi- role for Ni in other mechanisms of transmembrane signaling in donic acid is converted primarily to A2 that addition to receptor coupling to adenylate cyclase. Alternative- appears to be the proximate mediator of contraction (2). ly, a closely related guanine nucleotide binding protein, e.g., No In addition to their spasmogenic properties, LTC4 and (29), which is also recognized by IAP, may be involved. LTD4 also affect the vasculature by increasing permeability In the present study, IAP was used to probe the mechanism (4). Regulation of the vasculature is thought to be due to of signal transduction in CPAE cells. Previous studies dem- complex interactions between smooth muscle and endo- onstrated that LTD4- and LTC4-promoted prostacyclin syn- thelial cells (5). Via a process similar to that ofsmooth muscle thesis was dependent on both transcription and translation, cells, LTC4 and LTD4 enhance A2 activity in however, bradykinin-induced prostacyclin synthesis was the bovine endothelial cell line CPAE (3). However, in the under translational control (6). Here, we report that IAP endothelial cells, the primary product is specifically inhibits LTD4- and LTC4-, but not bradykinin-, prostacyclin (6). Because these cells produce prostacyclin in induced arachidonic acid release and subsequent prosta- response to LTD4, bradykinin, and the calcium ionophore cyclin synthesis consistent with the hypothesis that the A23187, this cell line provides an excellent model system for mechanisms of receptor coupling for the leukotrienes and elucidating the mechanism of prostacyclin induction. The bradykinin are distinctly different. physiological responses ofthe different cell lines appear to be MATERIALS AND METHODS determined by the major cyclooxygenase product synthe- Reagents. The LTD4 and LTC4 were prepared by total sized. Thus, some cells produce primarily contractile cyclo- synthesis and provided by J. Gleason (Medicinal Chemistry, oxygenase products and others, such as CPAE, produce Smith Kline and French Laboratories) and were greater than primarily the smooth muscle relaxant prostacyclin. Abbreviations: LTC4, leukotriene C4; LTD4, leukotriene D4; IAP, The publication costs of this article were defrayed in part by page charge islet-activating protein; Ni, guanine nucleotide regulatory protein payment. This article must therefore be hereby marked "advertisement" that mediates the inhibition of the cyclase enzyme; N., guanine in accordance with 18 U.S.C. §1734 solely to indicate this fact. nucleotide regulatory protein of unknown function. 7320 Downloaded by guest on September 30, 2021 Cell Biology: Clark et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7321

98% pure as determined by HPLC. The radioimmunoassays 10mM thymidine/1 mM ATP/0.5 mM GTP/5 MkM [32P]NAD/I were performed using kits obtained from New England AP at 5 Mg/ml (pretreated with 20 mM dithiothreitol for 10 Nuclear as described by the manufacturer. IAP was pur- min, 370C). At the end of the 20-min incubation, the mem- chased from List Biological Laboratories (Campbell, CA). branes were washed in buffer A and solubilized in NaDodSO4 [32P]NAD (10-50 Ci/mmol; 1 Ci = 37 GBq) and [3H]arachi- sample buffer (32). NaDodSO4/polyacrylamide gel electro- donic acid (50-60 Ci/mmol) were obtained from New En- phoresis was carried out as described by Laemmli (32). gland Nuclear. All other reagents were obtained from Sigma or Fisher. RESULTS Cell Culture. The bovine endothelial cell line, CPAE, was obtained from the American Type Culture Collection In the bovine endothelial cell line (CPAE), LTD4, LTC4, (Rockville, MD) and used within 10 passages of receipt. The bradykinin, and the calcium ionophore A23187 stimulate the cells were grown in Dulbecco's modified minimal essential synthesis of prostacyclin (6). Cellular responses to medium (GIBCO) supplemented with 20% (vol/vol) fetal calf leukotrienes appear to be receptor mediated (1, 7). Pretreat- serum (KC Biologicals, Kansas City, MO). All cells used in ment of cells with leukotriene antagonists, FPL55712 or these experiments were in the logarithmic phase of growth. SKF102081, completely inhibited prostacyclin synthesis in- None of the treatments described here decreased the cells' duced by 0.1 uM LTD4. viability as determined by trypan blue dye exclusion. To investigate the mechanisms by which these agents [3HjArachidonic Acid Metabolic Release. Cells were grown stimulate prostacyclin synthesis, we have examined the in 100-mm Petri dishes for 24-48 hr (approximately 50-70% effect of IAP on the endothelial cells. The production of confluent) prior to the addition of [3H]arachidonic acid (10 prostacyclin was determined by radioimmunoassay for 6- MCi/ml). Cells were incubated overnight at 370C, then rinsed keto-PGFi,, the stable breakdown product of prostacyclin three times with Puck's Saline F (GIBCO) containing 10 mM (Table 1). The CPAE cells were preincubated with and Hepes (pH 7.2). Fresh saline (10 ml) containing the stimulus without the toxin (100 ng/ml) for 1 hr prior to challenging was added to the dish. The dishes were then incubated at these cells with LTD4 and LTC4 (1 MM), bradykinin (2 ,uM), 370C, and 200-,ul aliquots of the culture supernatant were or A23187 (1 MLM)-concentrations that evoke a near maxi- removed in duplicate at the indicated times. IAP, when used, mal prostacyclin synthetic response (6). IAP had no effect on was added to the cell culture (100 ng/ml) 1 hr prior to the the basal production of this , however, IAP com- addition of the stimulus. The concentrations of these stimuli pletely inhibited the response to LTD4 and LTC4. The effect used provoke maximal responses (6), and the concentration of IAP appears to be specific to the LTD4- and LTC4- of bradykinin used in this report was similar to that employed dependent response because preexposure of CPAE cells to in studies on the effects of LAP (22, 30). However, we have the toxin had no significant effect on bradykinin- or also used bradykinin at a lower concentration (20 pM) with ionophore-stimulated prostacyclin production. identical results (data not shown). Analogous results were obtained when we examined ara- Radioimmunoassays for 6-Keto-Prostaglandin F1j. Cells chidonic acid release directly. In these experiments, CPAE were grown as described above in 24-well Linbro plates. The cells were incubated overnight in the presence of [3H]ara- cells were rinsed twice with saline and then incubated in 1 ml chidonic acid (10 MuCi/ml). The cells were then washed and of saline containing the stimulus for 10 min at 37°C. When treated with LTD4 and LTC4, bradykinin, or ionophore at the IAP was used, it was added to the cell culture (at the concentrations given above. Aliquots (200 Mil) were removed concentrations indicated) 1 hr prior to the experiment. The in duplicate at the indicated times, and the amount of amount of 6-keto-prostaglandin Fl, (6-keto-PGF1,) produced radioactivity released into the supernatants was determined was determined using radioimmunoassay kits as described by by scintillation spectrometry. The results are shown in Fig. the manufacturer. or did IAP, LTD4, LTC4, bradykinin not la. The magnitude of the responses was similar to that interfere with or cross-react with the antibody used in these reported in 3T3 fibroblasts (22). Although each of these radioimmunoassays. agents increased the release of radioactive material, the time Radioimmunoassays for cAMP. Cells were grown in 6-well period from initial challenge until stimulated release occurred plates until 80% confluent. The cells were rinsed twice with Dulbecco's modified minimal essential medium containing 10 was different. The ionophore A23187 increased the release of mM Hepes (pH 7.4) and 1 ,uM indomethacin. When used, LAP radioactive material very rapidly, in less than 1 min. After 2-4 (100 ng/ml) was added to the cell culture 1 hr prior to the min, LTD4 and LTC4 induced a statistically significant addition of the test compounds. Compounds were added in release of label. Bradykinin appeared to be intermediate in 1.0 ml of the above medium at the following final concentra- that -1 min was required for a significant increase in release tion: isoproterenol, 10 MM; forskolin, 50 MM; LTD4, 1 ,M; of labeled arachidonic acid metabolites. Although the lag bradykinin, 2 MuM. After a 10-min incubation at 37°C, cells periods preceding stimulated release of [3H]arachidonic acid were lysed by adding 100 ,ul of 100% trichloroacetic acid. Cell supernatants were extracted with ether to remove the tri- Table 1. The effect of IAP on prostacyclin synthesis in response chloroacetic acid, and cAMP was measured by radio- to bradykinin, ionophore A23187, LTC4, and LTD4 immunoassay as described by the manufacturer of the radio- 6-keto-PGFIa, ng/ml immunoassay kit. Membrane Labeling with [32P]NAD and LAP. Cell pellets Treatment Control IAP were first lysed by freezing and thawing (twice) and then None 2.4 ± 0.6 2.4 ± 0.2 resuspended in buffer A (20 mM Hepes/2 mM MgCl2/1 mM LTD4 (1 AM) 4.6 ± 0.2 2.5 ± 0.4* EDTA, pH 7.4). The cell lysates were homogenized by 10 LTC4 (1 ,M) 4.1 ± 0.4 1.9 ± 0.3* strokes in a Dounce homogenizer and then centrifuged at Bradykinin (2 uM) 4.3 ± 0.2 3.8 ± 0.4 1000 x g for 10 min. The resulting supernatants were Ionophore A23187 (1 MuM) 4.2 ± 0.1 3.9 ± 0.2t harvested and at x for 15 min. The centrifuged 40,000 g Cells were pretreated with LAP (100 ng/ml) for 1 hr prior to being resulting membrane pellets were resuspended in buffer A, treated with bradykinin (2 MAM), ionophore (1 uM), LTD4 (1 ,M), or and the protein concentrations were adjusted to 2 mg/ml. LTC4 (1 MM). After an additional 10-min incubation, the superna- Protein concentrations were determined by the method of tants were assayed for 6-keto-PGF1,, by radioimmunoassay. The data Bradford (31) using bovine y globulin as the standard. The shown was obtained from three experiments (mean ± SD). membranes (0.1 ml) were incubated for 20 min at 370C with *P < 0.001. tNot significant, P > 0.1. Downloaded by guest on September 30, 2021 7322 Cell Biology: Clark et al. Proc. Natl. Acad. Sci. USA 83 (1986)

U a a A I A 0 S 2 A 0

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.-I -43

C,,0 x A I II -30 a- 3 -b 0 lal 0 0 2 o Zs -20

20 0 8 1 2 3 4 5 6 0 1 A' ' ' ' 0.51 2 4 8 FIG. 2. [32P]ADP-ribosylation by 1AP of membranes prepared Time (min) from CPAE cells preincubated with various concentrations of IAP (100, 20, 10, 3, 0, and 0; lanes 1-6, respectively. CPAE cells were pretreated with the indicated concentrations of 1AP for 1 hr at 370C. FIG. 1. Release of [3H]arachidonic acid metabolites from CPAE Membranes were then prepared from the cells and subsequently cells pretreated in the absence (a) or presence (b) of LAP. CPAE cells incubated with 5 MM [32P]NAD in the presence (lanes 1-5) and were cultured overnight in the presence of [3H]arachidonic acid at 10 absence (lane 6) of preactivated 1AP at 5 ,Mg/ml. The membranes MCi/ml. The incubation was continued for an additional 1 hr in the were then dissolved in NaDodSO4 sample buffer. Electrophoresis absence (closed symbols) or presence (open symbols) of IAP at 100 was carried out on a 12-cm, 10% polyacrylamide gel. The gel was ng/ml. The monolayers of cells were rinsed twice using saline and dried and exposed to Kodak XAR-5 film in the presence of intensi- then challenged with 1 MiM LTD4 (o, a), 1 uM LTC4 (, o), 2 AM fying screens for 8 hr. The positions of molecular size standards in bradykinin (A, A), 1 uM ionophore (v, o), or saline alone (., o). kDa are indicated at the right. Aliquots from the supernatants (200 ,l) were removed in duplicate at the times indicated, and the radioactivity was determined. The results shown are the mean of three separate experiments. The phoresis. Endothelial cells preincubated with the same con- standard deviation is less than 10% for each data point. centrations of IAP were also assayed for prostacyclin pro- duction induced by 1 AM LTD4 or LTC4. Ifthe uncoupling of products were different, the total number of counts released the leukotriene-induced prostacyclin synthesis by IAP is into the supernatants after 8 min was the same independent related to the covalent modification of the 41-kDa membrane of the stimulus used. These data are similar to those obtained protein, pretreatment of the cells with various IAP concen- by radioimmunoassay for 6-keto-PGFia in these cells (6). trations should inhibit, in a dose-dependent manner, the Cells that had been preincubated with [3H]arachidonic acid incorporation of [32P]ADP-ribose into the substrate protein as in the above experiment were also pretreated with IAP during the subsequent membrane labeling experiment. As (100 ng/ml) for 1 hr prior to being stimulated with LTD4, can be seen in Fig. 3, this was observed, and a close LTC4, bradykinin, or ionophore. IAP had no detectable correlation exists between the inhibition of 32p labeling and effect on the basal release of radioactivity into the culture the inhibition of leukotriene-stimulated prostacyclin synthe- supernatants or on the release ofradioactive material induced sis. These data strongly suggest a role for the TAP substrate by either bradykinin or ionophore (Fig. lb). However, LTD4- in mediating LTD4 and LTC4 stimulation of prostacyclin and LTC4-induced release of radioactive material was com- synthesis in bovine endothelial cells. pletely inhibited by the toxin pretreatment. The molecular basis of TAP action appears to be a toxin- DISCUSSION catalyzed ADP-ribosylation of guanine nucleotide regulatory proteins (20, 28). IAP pretreatment of CPAE cells had no Leukotrienes contribute to and . effect on basal or forskolin-stimulated cAMP accumulation. Experiments using intact tissues and cultured cells have Moreover, cAMP accumulation in these cells was not affect- shown that leukotrienes increase prostanoid synthesis that ed by LTD4, LTC4, or bradykinin, and this observation was may play an important role in mediating these responses (2, unperturbed by toxin pretreatment (data not shown) suggest- 6). The toxin IAP has proven to be a useful tool to probe the ing that LTD4- and LTC4-induced prostacyclin synthesis and mechanisms of signal transduction in cells (20-28). The its inhibition by TAP are not related to cAMP . present studies show that IAP blocks LTD4- and LTC4- Exposure of membranes prepared from the endothelial induced arachidonic acid release and prostacyclin synthesis cells to [32P]NAD and preactivated IAP resulted in the in endothelial cells. It is unlikely that TAP was acting as an specific labeling of a 41-kDa protein in this preparation (Fig. LTD4 or LTC4 antagonist for at least two reasons. First, the 2). To determine if an TAP-promoted modification of this cells were pretreated with IAP, then washed extensively protein was related to the ability of the toxin to inhibit LTD4 prior to treatment with LTD4 or LTC4, thus there was little and LTC4 stimulation of prostacyclin synthesis, membranes free IAP present to compete with the leukotrienes for binding were prepared from CPAE cells that had been exposed to to their receptors. Second, simultaneous addition of IAP and various concentrations of TAP. These membranes were LTD4 or LTC4 did not inhibit prostacyclin synthesis and a subsequently radiolabeled in the presence of [32P]NAD and pretreatment time of at least 15 min was necessary to induce preactivated toxin and then solubilized in NaDodSO4, and inhibition of the leukotriene responses (data not shown). the samples were analyzed by polyacrylamide gel electro- Several investigators have also shown that a similar pretreat- Downloaded by guest on September 30, 2021 Cell Biology: Clark et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7323 A striking characteristic ofthe IAP effect on CPAE cells is the ability of the toxin to discriminate between the liberation 100 -A of arachidonic acid and prostacyclin synthesis induced by . LTD4 and LTC4 and that induced by bradykinin. IAP 0 pretreatment of the cells failed to inhibit arachidonic acid A release evoked by bradykinin at either 20 pM or 2 gM. That '5 . A IAP has no effect on the bradykinin-induced responses in 0 CPAE cells is in contrast to a report using 3T3 fibroblasts 0 0 (22). In fibroblasts, IAP inhibited bradykinin-induced arachi- a 50 0 donic acid release. These data suggest that the mechanisms of coupling between bradykinin receptors and arachidonic

0 acid release may be dependent on cell type. In studies using A CPAE cells, we postulated that LTD4 and LTC4 modulate prostanoid synthesis by a mechanism different from that for

0 bradykinin (6). LTD4 and LTC4 stimulation of prostacyclin production is blocked by either cycloheximide or actinomy- A cin D. In contrast, the effect of bradykinin was inhibited by 0 1 3 - cycloheximide, but was insensitive to actinomycin D. Mam- 10 30 100 malian cells appear to have at least two pathways for the 1AP concentration (ng/ml) liberation of arachidonic acid from phospholipids. The first is FIG. 3. Inhibition of LTD4- and LTC4-stimulated prostacyclin direct release by , and the second is indirect synthesis and [32P]ADP-ribosylation of 41-kDa membrane protein by the combined action of followed by following pretreatment of CPAE cells with various concentrations of lipase. The regulation of by other IAP. CPAE cells were preincubated with the indicated concentra- proteins such as lipocortin has been reported (34). It has been tions of IAP for 1 hr. Membranes were then prepared from the cells, proposed that additional proteins may be involved in modu- [32P]ADP-ribosylated in the presence of [32P]NAD and IAP, and lating phospholipase activities, e.g., guanine nucleotide reg- analyzed by NaDodSO4/polyacrylamide gel electrophoresis as de- ulatory proteins (21, 22, 33-37). The present data, using IAP, scribed in Fig. 2. Gels were sliced, and the radioactivity of the 41-kDa support this proposal and place the site ofIAP inhibition prior protein was determined by scintillation spectrometry (a). These data to arachidonic acid release. This would indicate that the IAP are plotted with data obtained by radioimmunoassays for 6-keto- substrate may be specifically involved in the regulation of PGF1,, produced by cells treated with 1 MM LTD4 (A) and 1 AM LTC4 (e). The radioimmunoassays were performed using cells that had some, but not all, phospholipases releasing substrate for been pretreated with various concentrations of IAP for 1 hr, then 1 eicosanoid synthesis. The results are consistent with the AM LTD4 was added to the cell cultures, and the supernatants were notion that the mechanisms of signal transduction of assayed 10 min later. The radioimmunoassay data were obtained leukotriene and bradykinin receptors leading to increased from three separate experiments, and the [32P]ADP-ribosylation data prostanoid synthesis by endothelial cells may be different. were obtained from two experiments. The physiologic significance of multiple pathways for inflam- matory responses to these stimuli remains to be determined. ment time is necessary for 1AP to block other receptor- mediated effects and is consistent with the proposed We wish to thank Drs. H. M. Sarau and G. K. Hogaboom for mech- critically reading the manuscript and Ms. D. Lavan for expert anism of action for the toxin (for review see ref. 33). secretarial assistance. The ability ofIAP to inhibit the LTD4- and LTC4-promoted responses appears to be independent of cyclic nucleotide 1. Clark, M. A., Cook, M., Mong, S., Hogaboom, G. K., Shorr, metabolism and yet is consistent with the observation that R., Stadel, J. & Crooke, S. T. (1984) Life Sci. 35, 441-448. binding properties of LTD4 receptors are modulated by 2. Clark, M. A., Cook, M., Mong, S. & Crooke, S. T. (1985) Eur. guanine nucleotides 19). The J. Pharmacol. 116, 207-220. (8, 18, correlation of the effect 3. Clark, M. A., Littlejohn, D., Conway, T. M., Mong, S., of the toxin on cellular prostanoid biosynthesis with the Steiner, S. & Crooke, S. T. (1986) J. Biol. Chem. 261, ADP-ribosylation of a 41-kDa membrane protein suggests 10713-10718. that a regulatory protein similar to N, or No is required for 4. Lewis, R. A., Drazen, J. M., Austen, K. F., Clark, D. A. & LTD4- and LTC4-promoted arachidonic acid release and Corey, E. J. (1980) Biochem. Biophys. Res. Commun. 96, subsequent prostacyclin synthesis; however, the identity of 271-277. the IAP substrate in these experiments remains to be defined. 5. Weinberg, C. B. & Bell, E. (1986) Science 213, 397-400. 6. Clark, M. A., Littlejohn, D., S. T. These data are analogous to Mong, & Crooke, S. (1985) observations reported using 31, 157-166. guinea pig in which IAP uncouples N-formyl 7. Mong, S., Wu, H., Clark, M. A., Gleason, J. G. & Crooke, peptide stimulation of arachidonic acid release from mem- S. T. (1986) J. Pharmacol. Exp. Ther., in press. branes and calcium gating (20,21). In these studies, and in the 8. Bruns, R., Thomsen, W. J. & Pugsley, T. A. (1983) Life Sci. data presented here, the capacity of the ionophore A23187 to 33, 645-653. stimulate arachidonic acid release was shown to be insensi- 9. Pong, S. S., DeHaven, R. N., Kuehl, F. A. & Egan, R. (1983) tive to TAP treatment, presumably due to J. Biol. Chem. 258, 9616-9619. the ability of the 10. Mong, S., Wu, H. L., Clark, M. A., Stadel, J. M., Gleason, ionophore to directly increase the intracellular calcium con- J. G. & Crooke, S. T. (1984) Prostaglandins 28, 805-822. centration. The toxin-induced inhibition of agonist promoted 11. Mong, S., Wu, H. L., Scott, M. O., Lewis, M. A., Clark, responses was also correlated with the ADP- M. A., Weichman, B. M., Kinzig, C. M., Gleason, J. G. & ribosylation of a 41-kDa membrane protein and only a single Crooke, S. T. (1985) J. Pharmacol. Exp. Ther. 234, 316-325. TAP substrate was detected (20, 21). The ability of the toxin 12. Hogaboom, G. K., Mong, S., Wu, H. L. & Crooke, S. T. (1983) Biochem. Biophys. Res. Commun. 116, 1136-1143. to uncouple the N-formyl peptide-dependent responses did 13. Hogaboom, G. K., Mong, S., Stadel, J. M. & Crooke, S. T. not appear to be mediated by alterations in the intracellular (1985) J. Pharmacol. Exp. Ther. 233, 686-693. concentration of cyclic AMP. These results were interpreted 14. Hogaboom, G. K., Mong, S., Stadel, J. M. & Crooke, S. T. as evidence that the 41-kDa protein was probably the a (1984) Mol. Pharmacol. 27, 236-245. subunit of Ni and that the Ni regulatory protein may be 15. Lewis, M. A., Mong, S., Vessella, R. L. & Crooke, S. T. involved in multiple mechanisms of signal transduction (21). (1986) Biochem. Pharmacol. 34, 4311-4317. Downloaded by guest on September 30, 2021 7324 Cell Biology: Clark et al. Proc. Nati. Acad. Sci. USA 83 (1986)

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