- Email: [email protected]
Evidence for G-Protein-Dependent and G-Protein-Independent Activation of Phospholipase D in Lymphocytes
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
229, 630–634 (1996)
1855
Evidence for G-Protein-Dependent and G-Protein-Independent Activation of Phospholipase D in Lymphocytes Yu-Zhang Cao, Padala V. Reddy, Lorraine M. Sordillo, George R. Hildenbrandt, and C. Channa Reddy1 Environmental Resources Research Institute and Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802 Received October 24, 1996 Previously we reported that tumor-promoting phorbol esters stimulate phospholipase D (PLD) independent of protein kinase C (PKC) activation in bovine lymph node lymphocytes. (Cao et al., Biochem. Biophys. Res. Commun. 171, 955–962, 1990; 217, 908–915, 1995). In the present study, we examined the effects of prostagladins (PGs), E2 , F2a , D2 , and H2 on PLD activity as measured by conversion of [1-14C] arachidonic acid-labeled phospholipids into phosphatidylethanol (PEt) in bovine lymph node lymphocytes. Prostaglandins stimulated the formation of PEt at an optimal concentration of 10 mM with relative stimulatory effect on the order of PGE2 ú PGF2a ú PGH2 ú PGD2 . The PGE2-stimulated formation of PEt was dosedependent in the range of 0.1 to 10 mM and was not inhibited by PKC inhibitors staurosporine and K252a. When both PGE2 and 12-0-tetradecanoylphorbol-13-acetate (TPA) were included, their effect on the PLD activation was additive. Furthermore, NaF, a G-protein activator, stimulated the PEt formation. Interestingly, the stimulatory effects of PGE2 and NaF were not additive; however, the formation of PEt by NaF and TPA was additive. These results suggest that similar to TPA, PGs increase PLD activity independent of PKC and the stimulation by PGs and TPA in lymphocytes may involve both G-protein-dependent and Gprotein-independent signaling pathways. q 1996 Academic Press, Inc.
Phospholipase D (EC 3.1.4.4.) (PLD) activation has been recognized as an important route of signal transduction in a number of cell types. It has been implicated in the regulation of DNA synthesis, cell proliferation and many other cellular functions (1). Several factors, like GTP-binding protein (G-protein), protein kinase C (PKC), protein-tyrosine kinase and calcium ions have been reported to regulate PLD activation. Phospholipase D activity was shown to be enhanced by the non-hydrolyzable GTP analogue GTP-g-S in a number of systems, including HL-60 cells (1), human neutrophils (2), dog brain microsomes (3), and rat liver plasma membranes (4). Recently, it was reported that prostaglandin (PG) F2a , an arachidonic acid metabolite, stimulates the activation of PLD in osteoblast-like cells(5) and in chinese hamster ovary (CHO) cells (6). In the previous studies (7,8), we demonstrated that the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) activates PLD via a PKC-independent mechanism in bovine lymph node lymphocytes. However, the precise mechanism(s) of PLD activation by TPA in immune cells have not been established. The present study was undertaken to delineate the molecular mechanisms of PLD activation by TPA as well as the role of PGs and NaF, a G-protein activator, in PLD activation in bovine lymphocytes. MATERIALS AND METHODS 14
Materials. [1- C] arachidonic acid ( specific activity 51.7 mCi/mmol) was purchased from Dupont NEN, Boston, MA. DMSO, TPA, PGE2 , PGD2 , PGF2a , staurosporine, NaF, and calcium ionophore A23187 were purchased from
1
Corresponding author. Fax: (814) 863-1696. E-mail: [email protected]. 630
0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
BBRC 5810
/
6913$$$901
11-22-96 06:40:10
bbrca
AP: BBRC
Vol. 229, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Relative effects of various agonists on PLD activity. Autoradiography of TLC separation of lipids extracted from cell cultures prelabeled with 1-14C arachidonic acid and incubated with various test compounds: (1) 0.1% DMSO; (2) 10 mM PGE2 ; (3) 100 nM TPA; (4) 0.2 mM 15-HPETE; (5) 40 mM NaF; (6) 1 mM Calcium ionophore A23187; (7) 3 mM Staurosporine / 100 nM TPA; (8) 1 mM K252a / 100 nM TPA. Incubation was for 3 h in the presence of 1% ethanol or 0.1% DMSO and indicated test compounds.
Sigma Chemical Co., St. Louis, MO. PGH2 was prepared in our laboratory as previously described (9). K252a was obtained from Kamiya Biochemical Co., Thousand Oaks, CA. RPMI-1640 with L-glutamine medium was purchased from Mediatech Co., Washington, DC. Precoated silica gel 150A plates were from Whatman Laboratory, Clifton, NJ. Preparation of bovine lymph node lymphocytes. Lymphocytes were prepared from bovine retropharyngeal lymph nodes and cultured as described previously (10). Cell cultures (108 cells/5 ml) were prelabeled with [1-14C] arachidonic acid (0.1 mCi/ml) at 377C for 1 h, washed once with RPMI-1640 medium and resuspended in fresh medium. Measurement of phospholipase D activity. Quantitation of radioactive phosphatidylethanol (PEt) formed from the endogenous prelabeled phospholipids in the presence of ethanol was used to measure PLD activity. All incubations on prelabeled cultures were conducted in presence of test compounds and 1.0 % ethanol for 3 h or as indicated in the legends of Figure and Tables. Test compounds were included in the 1.0 % ethanol additions except for TPA which was added in DMSO (0.1 % final concentration). In the experiments containing PKC inhibitor, the inhibitor was added 30 min prior to the addition of test compound and 1.0 % ethanol. The cells were centrifuged at 300 1 g for 10 min and washed once with phosphate-buffered saline (PBS) and the lipids were extracted. The lipid extracts were evaporated and applied to precoated silica gel 150A plates and the chromatograms developed with the organic phase of ethyl acetate-2,2,4-trimethylpentane-acetic acid-water (11:5:2:10, v/v/v/v). The lipids were visualized by iodine vapors and the radioactivity in PEt was determined by scraping the PEt band and subjecting it to liquid scintillation spectrometry as described before (7). Preparation of PEt standard. A PEt standard was synthesized using cabbage PLD and egg lecithin as described by Kobayashi and Kanfer (11). Data analysis. Data were analyzed by the Student’s t test and the statistical significance was assigned at p õ 0.05.
RESULTS
Effect of various agonists on PEt formation catalyzed by PLD. It is well recognized that the assay of PEt formation has many advantages over the assay of phosphatidic acid (PA) in the measurement of PLD activity. Double-labeling method demonstrated that PEt was formed from phospholipids exclusively by the action of PLD and it was metabolically stable, lasting up to 12 h after agonist treatment (12,13). Thus, in the present study, the PEt formation was measured to evaluate the activity of PLD in intact cells. As shown in Figure 1, TPA greatly stimulated PEt formation in lymphocytes. PGE2 and NaF also stimulated the PEt formation. As previously reported by us, the time course of PEt accumulation in TPA stimulated lymphocytes indicates that significant PEt accumulated within 15 min. The rate of increase remained relatively linear for 3 h. The increase in PEt with TPA was approximately 10 times greater than that with 0.1% DMSO alone with the highest stimulation of PEt synthesis seen at 1 1 1007 M TPA (7). The relative effects of different PGs on PLD-mediated PEt formation. As shown in Table 631
AID
BBRC 5810
/
6913$$$901
11-22-96 06:40:10
bbrca
AP: BBRC
Vol. 229, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1 Relative Effects of PGs on PLD-Mediated PEt Formation Test compounds
PEt formation (CPM)
Control PGE 2a PGF2aa PGD2a PGH2a
320 1123b 729b 469b 561b
{ { { { {
35 115 103 37 45
a
All incubations on prelabeled cultures were conducted in the presence of test compounds at 10 mM concentration and 1% ethanol for 3 h. b The values with a superscript are significantly different from the control value (p õ 0.05). Data are expressed as mean { S.D. for three individual experiments.
1, at 10 mM concentration, PGE2 , PGF2a , PGD2 , and PGH2 caused significant increase (põ0.05) in PLD activity in bovine lymph node lymphocytes with PGE2 showing the highest stimulatory effect followed by PGF2a ú PGH2 ú PGD2 . Time course of PGE2-stimulated PEt formation in bovine lymphocytes revealed that significant PEt accumulated within 1 h and the linear increase continued up to 3 h. A dose response study (not shown) revealed that PGE2 stimulated the formation of PEt in a concentration dependent manner in the range between 0.1-10 mM with the maximum stimulation of PEt formation achieved at 10 mM. At this concentration the PEt formation by PGE2 was increased approximately to 3 fold over the control (Table 1). The effect of PKC inhibitors on the activation of PLD by TPA and PGE2 . We examined the effects of two potent PKC inhibitors K252a and staurosporine on TPA- and PGE2-stimulated PEt formation in intact lymphocytes. These two compounds have been shown to be very selective in inhibiting PKC activity in different cells (14). Both K252a and staurosporine failed to affect PEt formation stimulated by TPA and PGE2 (Figure 1 and Table 2). These results strongly suggest that PKC is not involved in the activation of PLD by TPA and PGE2 in bovine lymph node lymphocytes. The comparative effects of PGE2 , NaF, and TPA on PEt formation. Sodium fluoride (NaF), a nonspecific activator of G-protein which has been widely used to activate G-protein (4,15), was employed to check its effect on activation of PLD. It was found that NaF at the concentration of 10-40 mM stimulates the formation of PEt in bovine lymphocytes with the maximum stimulation observed at 40 mM. At the maximum stimulatory concentration , PGE2 (10 mM) and NaF (40 mM) together did not result in additive effect (Table 3). However, the formation of PEt by a combination of 100 nM TPA and 40 mM NaF was additive. Furthermore, we found that the stimulation of PEt formation by TPA and by PGE2 at their maximum stimulatory concentration was additive (Table 3). DISCUSSION
It has been reported that arachidonic acid, but not other fatty acids, stimulates lymphocyte proliferation (16). Ecosanoids, enzymatic oxidation products of arachidonic acid, are formed by virtually every tissue in the body and play crucial roles in blood clotting, inflammation, control of vascular tone, renal function and reproductive functions (17). Moreover, lymphocytes were shown to respond and produce ecosanoids in a variety of 632
AID
BBRC 5810
/
6913$$$901
11-22-96 06:40:10
bbrca
AP: BBRC
Vol. 229, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2 The Effect of Protein Kinase C Inhibitors on the Activation of PLD Activity by TPA or PGE 2 Test compounds
Concentration
DMSO TPA K252a Staurosporine PGE 2 K252a /TPA Staurosporine /TPA K252a /PGE 2 Staurosporine /PGE 2
0.1% 1007 M 1006 M 3 1 1006 M 1005 M 1006 M 1007 M 3 1 1006 M 1007 M 1006 M 1005 M 3 1 1006 M 1005 M
PEt formation (CPM) 340 2540* 328 343 958*
{ { { { {
24 310 52 64 110
2348* { 228 2643* { 289 833* { 134 1072* { 115
Means with a superscript * are significantly different from the control (0.1% DMSO) value (p õ 0.05). In the last four treatments, K252a or Staurosporine was added 30 min before TPA or PGE 2 . The results are expressed as mean { S.D. (n Å 3).
disease conditions (18,19). However, the precise mechanism(s) by which eicosanoids affect these processes remains to be fully understood. In the present study, we examined the effect of PGs on PLD activity in bovine lymph node lymphocytes. Prostaglandins stimulated the formation of [1-14C]-labeled PEt at an optimal dose of 10 mM with relative stimulatory effect in the order of PGE2 ú PGF2a ú PGH2 ú PGD2 . The PGE2-stimulated formation of PEt was not inhibited by PKC inhibitors staurosporine and K252a. Although PGE 2 was reported to stimulate PLD in human erythroleukemia cells, other PGs including PGF 2a had no effect on PLD (20). Recently PGF2a was reported to activate PLD in Osteoblast-like cells (5). Our results in this study indicate that several PGs have the ability to activate
TABLE 3 Effect of TPA and NaF on the PGE 2-Induced Formation of PEt in Bovine Lymphocytes Test compounds
PEt formation (CPM)
Control (no addition) PGE 2 10 mM NaF 40 mM TPA 100 nM NaF 40 mM / PGE 2 10 mM TPA 100 nM / PGE 2 10 mM TPA 100 nM / NaF 40 mM
287 984 1210 2524 1236 3347 3466
{ { { { { { {
54a 116b 158b 225c 125b 336d 373d
Note. The pre-labeled cells were incubated with 1% ethanol, 10 mM PGE 2 , 40 mM NaF, 100 nM TPA, or combinations thereof as indicated. Each value represents the mean { S.D. of three individual experiments (n Å 3). Means with different superscript letters are significantly different from one another (p õ 0.05). 633
AID
BBRC 5810
/
6913$$$901
11-22-96 06:40:10
bbrca
AP: BBRC
Vol. 229, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PLD in lymphocytes. To our knowledge, this is the first report that shows PLD was stimulated by different PGs in one type of cells. We report here an activation of PLD by PGE2 which is independent of TPA with maximum stimulation of PEt formation by PGE2 being 13 of that observed for TPA. The effects of PGE2 and TPA on the formation of PEt were additive. Likewise the stimulatory effects of TPA and NaF were additive; however, the formation of PEt by NaF and PGE2 was not additive. In conclusion, we found that, similar to TPA, PGs activate PLD independent of PKC and the stimulation of PLD by PGs and TPA in lymphocytes may involve both G-protein-dependent and G-protein-independent signaling pathways. ACKNOWLEDGMENTS This research was supported by the National Institutes of Health Grants HL31245 and AI06347.
REFERENCES 1. Siddiqi, A. R., Smith, J. L., Ross, A. H., Qiu, R-G., Symons, M., and Exton, J. H. (1995) J. Biol. Chem. 270, 8466–8473. 2. Olson, S. C., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236–17242. 3. Qian, Z., Reddy, P. V., and Drewes, L. R. (1990) J. Neurochem. 54, 1632–1638. 4. Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987) J. Biol. Chem. 262, 15309–15315. 5. Kozawa, O., Suzuki, A., Kotoyori, J., Tokuda, H., Watanabe, Y., Ito, Y., and Oiso, Y. F. (1994) J. Cell. Biochem. 55, 373–379. 6. Liu, B., Nakashima, S., Ito, S., and Nozawa, Y. (1996) Prostaglandins 51, 233–248. 7. Cao, Y-Z., Reddy, C. C., and Mastro, A. M. (1990) Biochem. Biophys. Res. Commun. 171, 955–962. 8. Cao, Y-Z., Mastro, A. M., Eskew, M. L., Hildenbrandt, G., Reddy, P. V., and Reddy, C. C. (1995) Biochem. Biophys. Res. Commun. 217, 908–915. 9. Hong, Y., Li, C-H., Burgess, J. R., Chang, M., Salem, A., Srikumar, K., and Reddy, C. C. (1989) J. Biol. Chem. 264, 13793–13800. 10. Mastro, A. M., and Mueller, G. C. (1974) Exp. Cell Res. 88, 40–46. 11. Kobayashi, M., and Kanfer, J. N. (1987) J. Neurochem. 48, 1597–1603. 12. Pei, J.-K., Siegel, M. I., Egan, R. W., and Billah, M. M. (1988) J. Biol. Chem. 263, 12472–12477. 13. Tettenborn, C. S., and Mueller, G. C. (1987) Biochim. Biophys. Acta 931, 242–250. 14. Davis, P. D., Hill, C. H., Keech, E., Lawton, G., Nixon, J. S., Sedgwick, A. D., Wadsworth, J., Westmacott, D., and Wilkinson, S. E. (1989) FEBS Letters 259, 61–63. 15. Gilman, A. G. (1986) Annu. Rev. Biochem. 56, 615–649. 16. Kelly, J. P., and Parker, C. W. (1979) J. Immunol. 122, 1556–1562. 17. Shimizu, T., and Wolfe, L. S. (1990) J. Neurochem. 55, 1–15. 18. Odlander, B., Jakobsson, P. J., Medina, J. F., Redmark, O., Yamaoka, K. A., Rosen, A., and Claesson, H-E. (1989) Int. J. Tissue React. 11, 277–289. 19. Cao, Y-Z., Maddox, J. F., Mastro, A. M., Scholz, R. W., Hildenbrandt, G., and Reddy, C. C. (1992) J. Nutr. 122, 2121–2127. 20. Wu, H., Turner, J. T., and Halenda, S. P. (1991) J. Pharmacol. Exp. Ther. 258, 607–612.
634
AID
BBRC 5810
/
6913$$$901
11-22-96 06:40:10
bbrca
AP: BBRC
229, 630–634 (1996)
1855
Evidence for G-Protein-Dependent and G-Protein-Independent Activation of Phospholipase D in Lymphocytes Yu-Zhang Cao, Padala V. Reddy, Lorraine M. Sordillo, George R. Hildenbrandt, and C. Channa Reddy1 Environmental Resources Research Institute and Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802 Received October 24, 1996 Previously we reported that tumor-promoting phorbol esters stimulate phospholipase D (PLD) independent of protein kinase C (PKC) activation in bovine lymph node lymphocytes. (Cao et al., Biochem. Biophys. Res. Commun. 171, 955–962, 1990; 217, 908–915, 1995). In the present study, we examined the effects of prostagladins (PGs), E2 , F2a , D2 , and H2 on PLD activity as measured by conversion of [1-14C] arachidonic acid-labeled phospholipids into phosphatidylethanol (PEt) in bovine lymph node lymphocytes. Prostaglandins stimulated the formation of PEt at an optimal concentration of 10 mM with relative stimulatory effect on the order of PGE2 ú PGF2a ú PGH2 ú PGD2 . The PGE2-stimulated formation of PEt was dosedependent in the range of 0.1 to 10 mM and was not inhibited by PKC inhibitors staurosporine and K252a. When both PGE2 and 12-0-tetradecanoylphorbol-13-acetate (TPA) were included, their effect on the PLD activation was additive. Furthermore, NaF, a G-protein activator, stimulated the PEt formation. Interestingly, the stimulatory effects of PGE2 and NaF were not additive; however, the formation of PEt by NaF and TPA was additive. These results suggest that similar to TPA, PGs increase PLD activity independent of PKC and the stimulation by PGs and TPA in lymphocytes may involve both G-protein-dependent and Gprotein-independent signaling pathways. q 1996 Academic Press, Inc.
Phospholipase D (EC 3.1.4.4.) (PLD) activation has been recognized as an important route of signal transduction in a number of cell types. It has been implicated in the regulation of DNA synthesis, cell proliferation and many other cellular functions (1). Several factors, like GTP-binding protein (G-protein), protein kinase C (PKC), protein-tyrosine kinase and calcium ions have been reported to regulate PLD activation. Phospholipase D activity was shown to be enhanced by the non-hydrolyzable GTP analogue GTP-g-S in a number of systems, including HL-60 cells (1), human neutrophils (2), dog brain microsomes (3), and rat liver plasma membranes (4). Recently, it was reported that prostaglandin (PG) F2a , an arachidonic acid metabolite, stimulates the activation of PLD in osteoblast-like cells(5) and in chinese hamster ovary (CHO) cells (6). In the previous studies (7,8), we demonstrated that the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) activates PLD via a PKC-independent mechanism in bovine lymph node lymphocytes. However, the precise mechanism(s) of PLD activation by TPA in immune cells have not been established. The present study was undertaken to delineate the molecular mechanisms of PLD activation by TPA as well as the role of PGs and NaF, a G-protein activator, in PLD activation in bovine lymphocytes. MATERIALS AND METHODS 14
Materials. [1- C] arachidonic acid ( specific activity 51.7 mCi/mmol) was purchased from Dupont NEN, Boston, MA. DMSO, TPA, PGE2 , PGD2 , PGF2a , staurosporine, NaF, and calcium ionophore A23187 were purchased from
1
Corresponding author. Fax: (814) 863-1696. E-mail: [email protected]. 630
0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
BBRC 5810
/
6913$$$901
11-22-96 06:40:10
bbrca
AP: BBRC
Vol. 229, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Relative effects of various agonists on PLD activity. Autoradiography of TLC separation of lipids extracted from cell cultures prelabeled with 1-14C arachidonic acid and incubated with various test compounds: (1) 0.1% DMSO; (2) 10 mM PGE2 ; (3) 100 nM TPA; (4) 0.2 mM 15-HPETE; (5) 40 mM NaF; (6) 1 mM Calcium ionophore A23187; (7) 3 mM Staurosporine / 100 nM TPA; (8) 1 mM K252a / 100 nM TPA. Incubation was for 3 h in the presence of 1% ethanol or 0.1% DMSO and indicated test compounds.
Sigma Chemical Co., St. Louis, MO. PGH2 was prepared in our laboratory as previously described (9). K252a was obtained from Kamiya Biochemical Co., Thousand Oaks, CA. RPMI-1640 with L-glutamine medium was purchased from Mediatech Co., Washington, DC. Precoated silica gel 150A plates were from Whatman Laboratory, Clifton, NJ. Preparation of bovine lymph node lymphocytes. Lymphocytes were prepared from bovine retropharyngeal lymph nodes and cultured as described previously (10). Cell cultures (108 cells/5 ml) were prelabeled with [1-14C] arachidonic acid (0.1 mCi/ml) at 377C for 1 h, washed once with RPMI-1640 medium and resuspended in fresh medium. Measurement of phospholipase D activity. Quantitation of radioactive phosphatidylethanol (PEt) formed from the endogenous prelabeled phospholipids in the presence of ethanol was used to measure PLD activity. All incubations on prelabeled cultures were conducted in presence of test compounds and 1.0 % ethanol for 3 h or as indicated in the legends of Figure and Tables. Test compounds were included in the 1.0 % ethanol additions except for TPA which was added in DMSO (0.1 % final concentration). In the experiments containing PKC inhibitor, the inhibitor was added 30 min prior to the addition of test compound and 1.0 % ethanol. The cells were centrifuged at 300 1 g for 10 min and washed once with phosphate-buffered saline (PBS) and the lipids were extracted. The lipid extracts were evaporated and applied to precoated silica gel 150A plates and the chromatograms developed with the organic phase of ethyl acetate-2,2,4-trimethylpentane-acetic acid-water (11:5:2:10, v/v/v/v). The lipids were visualized by iodine vapors and the radioactivity in PEt was determined by scraping the PEt band and subjecting it to liquid scintillation spectrometry as described before (7). Preparation of PEt standard. A PEt standard was synthesized using cabbage PLD and egg lecithin as described by Kobayashi and Kanfer (11). Data analysis. Data were analyzed by the Student’s t test and the statistical significance was assigned at p õ 0.05.
RESULTS
Effect of various agonists on PEt formation catalyzed by PLD. It is well recognized that the assay of PEt formation has many advantages over the assay of phosphatidic acid (PA) in the measurement of PLD activity. Double-labeling method demonstrated that PEt was formed from phospholipids exclusively by the action of PLD and it was metabolically stable, lasting up to 12 h after agonist treatment (12,13). Thus, in the present study, the PEt formation was measured to evaluate the activity of PLD in intact cells. As shown in Figure 1, TPA greatly stimulated PEt formation in lymphocytes. PGE2 and NaF also stimulated the PEt formation. As previously reported by us, the time course of PEt accumulation in TPA stimulated lymphocytes indicates that significant PEt accumulated within 15 min. The rate of increase remained relatively linear for 3 h. The increase in PEt with TPA was approximately 10 times greater than that with 0.1% DMSO alone with the highest stimulation of PEt synthesis seen at 1 1 1007 M TPA (7). The relative effects of different PGs on PLD-mediated PEt formation. As shown in Table 631
AID
BBRC 5810
/
6913$$$901
11-22-96 06:40:10
bbrca
AP: BBRC
Vol. 229, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1 Relative Effects of PGs on PLD-Mediated PEt Formation Test compounds
PEt formation (CPM)
Control PGE 2a PGF2aa PGD2a PGH2a
320 1123b 729b 469b 561b
{ { { { {
35 115 103 37 45
a
All incubations on prelabeled cultures were conducted in the presence of test compounds at 10 mM concentration and 1% ethanol for 3 h. b The values with a superscript are significantly different from the control value (p õ 0.05). Data are expressed as mean { S.D. for three individual experiments.
1, at 10 mM concentration, PGE2 , PGF2a , PGD2 , and PGH2 caused significant increase (põ0.05) in PLD activity in bovine lymph node lymphocytes with PGE2 showing the highest stimulatory effect followed by PGF2a ú PGH2 ú PGD2 . Time course of PGE2-stimulated PEt formation in bovine lymphocytes revealed that significant PEt accumulated within 1 h and the linear increase continued up to 3 h. A dose response study (not shown) revealed that PGE2 stimulated the formation of PEt in a concentration dependent manner in the range between 0.1-10 mM with the maximum stimulation of PEt formation achieved at 10 mM. At this concentration the PEt formation by PGE2 was increased approximately to 3 fold over the control (Table 1). The effect of PKC inhibitors on the activation of PLD by TPA and PGE2 . We examined the effects of two potent PKC inhibitors K252a and staurosporine on TPA- and PGE2-stimulated PEt formation in intact lymphocytes. These two compounds have been shown to be very selective in inhibiting PKC activity in different cells (14). Both K252a and staurosporine failed to affect PEt formation stimulated by TPA and PGE2 (Figure 1 and Table 2). These results strongly suggest that PKC is not involved in the activation of PLD by TPA and PGE2 in bovine lymph node lymphocytes. The comparative effects of PGE2 , NaF, and TPA on PEt formation. Sodium fluoride (NaF), a nonspecific activator of G-protein which has been widely used to activate G-protein (4,15), was employed to check its effect on activation of PLD. It was found that NaF at the concentration of 10-40 mM stimulates the formation of PEt in bovine lymphocytes with the maximum stimulation observed at 40 mM. At the maximum stimulatory concentration , PGE2 (10 mM) and NaF (40 mM) together did not result in additive effect (Table 3). However, the formation of PEt by a combination of 100 nM TPA and 40 mM NaF was additive. Furthermore, we found that the stimulation of PEt formation by TPA and by PGE2 at their maximum stimulatory concentration was additive (Table 3). DISCUSSION
It has been reported that arachidonic acid, but not other fatty acids, stimulates lymphocyte proliferation (16). Ecosanoids, enzymatic oxidation products of arachidonic acid, are formed by virtually every tissue in the body and play crucial roles in blood clotting, inflammation, control of vascular tone, renal function and reproductive functions (17). Moreover, lymphocytes were shown to respond and produce ecosanoids in a variety of 632
AID
BBRC 5810
/
6913$$$901
11-22-96 06:40:10
bbrca
AP: BBRC
Vol. 229, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2 The Effect of Protein Kinase C Inhibitors on the Activation of PLD Activity by TPA or PGE 2 Test compounds
Concentration
DMSO TPA K252a Staurosporine PGE 2 K252a /TPA Staurosporine /TPA K252a /PGE 2 Staurosporine /PGE 2
0.1% 1007 M 1006 M 3 1 1006 M 1005 M 1006 M 1007 M 3 1 1006 M 1007 M 1006 M 1005 M 3 1 1006 M 1005 M
PEt formation (CPM) 340 2540* 328 343 958*
{ { { { {
24 310 52 64 110
2348* { 228 2643* { 289 833* { 134 1072* { 115
Means with a superscript * are significantly different from the control (0.1% DMSO) value (p õ 0.05). In the last four treatments, K252a or Staurosporine was added 30 min before TPA or PGE 2 . The results are expressed as mean { S.D. (n Å 3).
disease conditions (18,19). However, the precise mechanism(s) by which eicosanoids affect these processes remains to be fully understood. In the present study, we examined the effect of PGs on PLD activity in bovine lymph node lymphocytes. Prostaglandins stimulated the formation of [1-14C]-labeled PEt at an optimal dose of 10 mM with relative stimulatory effect in the order of PGE2 ú PGF2a ú PGH2 ú PGD2 . The PGE2-stimulated formation of PEt was not inhibited by PKC inhibitors staurosporine and K252a. Although PGE 2 was reported to stimulate PLD in human erythroleukemia cells, other PGs including PGF 2a had no effect on PLD (20). Recently PGF2a was reported to activate PLD in Osteoblast-like cells (5). Our results in this study indicate that several PGs have the ability to activate
TABLE 3 Effect of TPA and NaF on the PGE 2-Induced Formation of PEt in Bovine Lymphocytes Test compounds
PEt formation (CPM)
Control (no addition) PGE 2 10 mM NaF 40 mM TPA 100 nM NaF 40 mM / PGE 2 10 mM TPA 100 nM / PGE 2 10 mM TPA 100 nM / NaF 40 mM
287 984 1210 2524 1236 3347 3466
{ { { { { { {
54a 116b 158b 225c 125b 336d 373d
Note. The pre-labeled cells were incubated with 1% ethanol, 10 mM PGE 2 , 40 mM NaF, 100 nM TPA, or combinations thereof as indicated. Each value represents the mean { S.D. of three individual experiments (n Å 3). Means with different superscript letters are significantly different from one another (p õ 0.05). 633
AID
BBRC 5810
/
6913$$$901
11-22-96 06:40:10
bbrca
AP: BBRC
Vol. 229, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PLD in lymphocytes. To our knowledge, this is the first report that shows PLD was stimulated by different PGs in one type of cells. We report here an activation of PLD by PGE2 which is independent of TPA with maximum stimulation of PEt formation by PGE2 being 13 of that observed for TPA. The effects of PGE2 and TPA on the formation of PEt were additive. Likewise the stimulatory effects of TPA and NaF were additive; however, the formation of PEt by NaF and PGE2 was not additive. In conclusion, we found that, similar to TPA, PGs activate PLD independent of PKC and the stimulation of PLD by PGs and TPA in lymphocytes may involve both G-protein-dependent and G-protein-independent signaling pathways. ACKNOWLEDGMENTS This research was supported by the National Institutes of Health Grants HL31245 and AI06347.
REFERENCES 1. Siddiqi, A. R., Smith, J. L., Ross, A. H., Qiu, R-G., Symons, M., and Exton, J. H. (1995) J. Biol. Chem. 270, 8466–8473. 2. Olson, S. C., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236–17242. 3. Qian, Z., Reddy, P. V., and Drewes, L. R. (1990) J. Neurochem. 54, 1632–1638. 4. Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987) J. Biol. Chem. 262, 15309–15315. 5. Kozawa, O., Suzuki, A., Kotoyori, J., Tokuda, H., Watanabe, Y., Ito, Y., and Oiso, Y. F. (1994) J. Cell. Biochem. 55, 373–379. 6. Liu, B., Nakashima, S., Ito, S., and Nozawa, Y. (1996) Prostaglandins 51, 233–248. 7. Cao, Y-Z., Reddy, C. C., and Mastro, A. M. (1990) Biochem. Biophys. Res. Commun. 171, 955–962. 8. Cao, Y-Z., Mastro, A. M., Eskew, M. L., Hildenbrandt, G., Reddy, P. V., and Reddy, C. C. (1995) Biochem. Biophys. Res. Commun. 217, 908–915. 9. Hong, Y., Li, C-H., Burgess, J. R., Chang, M., Salem, A., Srikumar, K., and Reddy, C. C. (1989) J. Biol. Chem. 264, 13793–13800. 10. Mastro, A. M., and Mueller, G. C. (1974) Exp. Cell Res. 88, 40–46. 11. Kobayashi, M., and Kanfer, J. N. (1987) J. Neurochem. 48, 1597–1603. 12. Pei, J.-K., Siegel, M. I., Egan, R. W., and Billah, M. M. (1988) J. Biol. Chem. 263, 12472–12477. 13. Tettenborn, C. S., and Mueller, G. C. (1987) Biochim. Biophys. Acta 931, 242–250. 14. Davis, P. D., Hill, C. H., Keech, E., Lawton, G., Nixon, J. S., Sedgwick, A. D., Wadsworth, J., Westmacott, D., and Wilkinson, S. E. (1989) FEBS Letters 259, 61–63. 15. Gilman, A. G. (1986) Annu. Rev. Biochem. 56, 615–649. 16. Kelly, J. P., and Parker, C. W. (1979) J. Immunol. 122, 1556–1562. 17. Shimizu, T., and Wolfe, L. S. (1990) J. Neurochem. 55, 1–15. 18. Odlander, B., Jakobsson, P. J., Medina, J. F., Redmark, O., Yamaoka, K. A., Rosen, A., and Claesson, H-E. (1989) Int. J. Tissue React. 11, 277–289. 19. Cao, Y-Z., Maddox, J. F., Mastro, A. M., Scholz, R. W., Hildenbrandt, G., and Reddy, C. C. (1992) J. Nutr. 122, 2121–2127. 20. Wu, H., Turner, J. T., and Halenda, S. P. (1991) J. Pharmacol. Exp. Ther. 258, 607–612.
634
AID
BBRC 5810
/
6913$$$901
11-22-96 06:40:10
bbrca
AP: BBRC