Phorbol Myristate Acetate Enhances Adenylate Cyclase Activity in Human Polymorphonuclear Leukocytes C.Rebut-Bonneton1, J. Demignon1 and P. Sourbier2 U262INSERM 2 U 294INSERM and Laboratoire d'Hematologic et d'Immunologie CHU Xavier-Bichat Universite Paris, Paris, France
Summary The putative role of protein phosphorylation in modulating adenylate cyclase activity in polymorphonuclear neutrophil membranes was assessed using phorbol myristate acetate (PMA) to stimulate the activity of protein kinase C. PMA was demonstrated to enhance the adenylate cyclase activity stimulated by isoproterenol. Key words Adenylate Cyclase - Polymorphonuclear Leukocytes - Isoproterenol - Protein Kinase C - Formyl Methionyl - Leucyl- Phenylalanine
To determine whether PKc changes the response of polymorphonuclear leukocytes (PMN) to isoproterenol, intact PMN, 40000 g-crude membranes or the membranes plus supernatant were treated with phorbol myristate acetate (PMA) before incubation with isoproterenol. PMA was previously shown to induce PKc translocation from the cytosol to the membrane (Wolfson, McPhail, Nasrallah and Snyderman 1985) where PKc was assumed to phosphorylate subunits of the adenylate cyclase system. Adenylate cyclase activity was measured by the rate of ATP conversion into cAMP, using a radioimmunoassay. Materials and Methods Material Phorbol myristate acetate (PMA), N formylmethionyl-leucyl-phenylalanine (FMLP), isoproterenol, isobutylmethylxanthine (IBMX), ATP, GTP, creatine phosphokinase and phosphocreatine were obtained from Sigma. The radioimmunoassay kit for cAMP determination by acetylation was purchased from New England Nuclear.
Introduction Cyclic adenosine monophosphate (cAMP) has long been recognized as the second messenger in many hormonal actions as well as in the control of cell proliferation. The molecular mechanism regulating the enzyme responsible for cAMP synthesis, i. e. adenylate cyclase (Ac), is now well characterized (Gilman 1984) and is known to involve a complex coupling system with different stimulatory or inhibitory receptors, and corresponding GTP binding proteins. The intracellular concentration of cAMP is also regulated by phosphodiesterase. Recent reports suggest that protein kinase C (PKc) is able to modify a component of adenylate cyclase, thus inducing changes in cell cAMP content. Fibroblasts (RochetteEgly and Castagna 1979) and epidermal cells (Grim and Marks 1974) exposed to the phorbol ester tumor promotor 12-0-tetradecanoyl phorbol-13-acetate (TPA) exhibited reduced ability to accumulate cAMP in response to hormones. This phorbol ester also inhibited isoproterenol-stimulated adenylate cyclase in human peripheral blood mononuclear cells (Novogrosky Patya, Rubin and Stenzel 1982).
Horm. metabol. Res. 22 (1990) 175-178 © Georg Thieme Verlag Stuttgart • New York
Adenylate cyclase assays in preparations
membranous
Venous blood was obtained on heparin (10 U/ml) from healthy adult donors. Purified PMN preparations were isolated from it, using Ficoll/Hypaque gradients (Mono-poly resolving medium, Flow Laboratories) and erythrocytes were lysed in hypotonic medium. Cells were suspended in an ice-cold buffered solution pH 7.4 (KRP) containing, in mM, 138 NaCl, 2.7 KC1, 8.1 Na2HPO4, 1.5 K2HPO4 and 1.0 MgCl2. Intact PMN were incubated at 37 °C for the indicated time in KRP solution containing 0.01 mM Ca 2+ (20 • 106 cells/ml) in the presence or absence of PMA. They were then quickly transferred to an ice bath and centrifuged for 8 min at 4 °C and 600 g. The pellet was diluted to reach 40 • 10 cells perml in bufferA(pH7.7) containing in mM, 50 Tris HC1, 10 MgCl2 and 2 EDTA. The suspension was homogenized three times for 30 sec each with a Potter homogenizer and centrifuged for 8 min at 200 g to remove nuclei and large debris. The resulting supernatant was centrifuged at 41000 g for 12 min and the remaining pellet containing the cell membranes and subcellular particles was resuspended by 10 sec sonication in 500 |xl of buffer B (pH 7.5) containing 75 mM Tris HC1,25 mMMgCl2and 10-4 M IBMX, and stored on ice. The protein concentration in the pellet
Received: 6 March 1989
Accepted: 31 July 1989
Downloaded by: Boston University. Copyrighted material.
1
Horm. metabol. Res. 22 (1990)
C. Rebut-Bonneton, J. Demignon and P. Sourbier
was adjusted to 6 ug/20 ul by dilution in buffer B, and the protein concentration in the supernatant was adjusted to 2 ug/20 ul. A reaction mixture was prepared on ice by sequentially adding 20 ul of membrane preparation, and/or 10 ul of supernatant and 10 ul of stimulant. The preliminary incubation with PMA was performed for 5 min at 0 °C; afterwards, isoproterenol was added to the final mixture containing 0.1 mM ATP, 0.1 mM GTP, 5 mM phosphocreatine and 0.1 mg/ml of creatine phosphokinase (phosphocreatine and creatine phosphokinase were used to generate ATP). Ac activation was initiated by placing the tubes in a 37 C water bath for 10 min and was stopped by icing the tubes. Accumulated cAMP was measured by radioimmunoassay using a NEN kit, and not by the usual procedure designed to evaluate the production of labelled cAMP from the 32P-ATP substrate (Solomon, Londos and Rodbell 1974) 200 ul of ice-cold acetate buffer and 5 ul of acetylation reagent, prepared by mixing 100 ul of triethylamine and 50 ul of acetic anhydride in a glass tube, were added. Three minutes were allowed for
acetylation at room temperature, and 100 ul of each sample was then transferred into plastic assay tubes. 100 ulof ,25I-cAMPand 100 ulof antiserum complex were then added to these tubes as indicated in the kit for cAMP determination. Results As shown by the data in Table 1,10 - 4 or 10~5 M isoproterenol significantly stimulated adenylate cyclase activity of membrane preparations, thus raising cAMP levels.
Table 1 Effect on PMN adenylate cyclase activity of pretreating PMN membranes with 10 ng/ml PMA for 10 min before their stimulation by isoproterenol. The reaction mixture contained 0.1 mM ATP, 0.1 mM GTP, 5 mM creatine phsophate, and 0.1 mg/ml creatine phosphokinase. cAMP accumulation was measured by radioimmunoassay. Control
100 ± 1 4 (3)
Isoproterenol 10- 5 M
237 ± 4 9 * * * (3)
Isoproterenol 10- 4 M
236 ± 19*** (3)
10 ng/ml PMA + 10-5 M isoproterenol
250 ± 2 2 * * * (3)
100ng/mlPMA + 10 M isoproterenol
207 ± 2 9 * * * (3)
Fig. 1 Effect of pretreatment of the supernatant + crude membrane preparation on adenylate cyclase activation by isoproterenol. cAMP accumulation was measured by radioimmunoassay. The "generator" of ATP was a mixture containing 0.1 mM ATP, 5 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase and 0.1 mM GTP. 1: membrane + supernatant 2: membrane + supernatant + 10~5 M isoproterenol 3,4: membrane + supernatant + PMA (10and 10 ng/ml respectively) + 10 _5 M isoproterenol. The mean control value was taken as 100 and the other values were calculated on this basis. Results are means ± SD. a P < 0.01 in comparison with control value b P < 0.01 in comparison with isoproterenol alone (variance analysis)
The mean control value was taken as 100 and the other values were calculated on this basis. Number of experiments is in parenthesis. ***P < 0.01 (variance analysis) versus to the control.
Table 2 Effect on the adenylate cyclase activity in PMN membranes of treating intact PMN with PMA for 5 min at 37 °C before incubating the membranes for 10 min at 37 °C with 10~5 M isoproterenol. PMN membranes from intact PMN treated with PMA for 5 min (ng/ml)
Control PMN membranes
Control
Isoproterenol 10-5M
8.99 ± 1.60 (3)
*** 15.40 ±1.83 (3)
0.1
1
10
11.90 ±3.64 (3)
10.31 ± 1.15 (6)
9.94 ±1.15 (6)
*** 18.90 ±1.83 (3)
***
22.07 ± 4.68ooc) (6)
* * +
26.84 ± 5.21 °°° (6)
cAMP accumulation in the presence of a reaction mixture containing 0.1 mM ATP, 0.1 mM GTP, 5 mM creatine phosphate and 0.1 mg/ml creatine phosphokinase. cAMP was measured by radioimmunoassay. Results are expressed in fmoles/min • |ig membrane protein (means ± SD). * * *P < 0.01 versus to the corresponding control """variance analysis of pretreated versus control PMN () number of experiments is in parenthesis
Downloaded by: Boston University. Copyrighted material.
176
Adenylate Cyclase in PMN
Horm. metabol. Res. 22 (1990)
Pretreatment of plasma membranes for 10 min with 10 or 100 ng/ml PMA did not enhance the degree of adenylate cyclase activation by these isoproterenol concentrations. The supernatant alone did not show any detectable Ac activity, and the addition of supernatant to the membranous preparation did not alter the response of Ac to isoproterenol; pretreatment of the membrane + supernatant mixture with PMA for 5 min at 0 °C before adding isoproterenol for Ac activation enhanced this activation (Fig. 1).
Becker and Sha'agi 1984). Consequently, this agent probably exerts its effect through the production of intermediate agents and/or requires substances available in intact cells but not in membrane preparations. Here, PKc should have been the intermediate agent but the pretreatment of intact PMN by PMA did not permit subsequent incubation with FMLP to raise Ac activity (Rebut-Bonneton, Demignon and Sourbier1988).However according to Verghese, Smith and Snyderman (1988), the chemoattractant might be coupled to an alternative effector mechanism via an N protein.
Pretreatment of intact PMN for 5 min at 37 °C with PMA also enhanced the Ac activation by isoproterenol in plasma membranes (Table 2). After incubation with isoproterenol a positive correlation was found between the concentration of PMA in the incubation medium and the adenylate cyclase activity in the membrane (r = 0.702, P < 0.01). PMA alone did not exert any detectable stimulation.
In conclusion, in PMN, PMA was shown to be an agent responsible for modulating the cAMP level through the PKc stimulation. In some cases, the effect of PMA could help in discriminating between a cell content cAMP increase due to a direct action on the adenylate cyclase system or to another mechanism. Acknowledgements This work was carried out in U 294 INSERM, 46 rue
The fact that in these membrane preparations Huchard, 750 18 Paris. ATP was converted into cAMP through hormone stimulation, References and also through the non specific agent NaF (result not shown) demonstrates that the catalytic unit of Ac remained inBeckner, S. K., W. L. Farrar: Inhibition of adenylate cyclase by IL2 in tact and functional. The presence of guanine nucleotides was human T lymphocytes is mediated by protein kinase C. Biochem. an absolute requirement for Ac activation by isoproterenol but Biophys. Res. Commun. 145:176-181 (1987) was not necessary for its activation by NaF stimulation. Iso- Bell, J. D., I. Buxton, L. L. Burton: Enhancement of adenylate cyclase proterenol was previously shown to activate Ac (Tecoma, activity in S49 lymphoma cells by phorbol ester: putative effect of Matulsky, Traynor, Omann, Mutter and Sklar 1986). Here, C kinase on Ls-GTP catalytic subunit interaction. J. Biol. Chem. PMA was demonstrated to reinforce the stimulation of Ac ac280:2625-2628(1985) Gilman, A. G.: G proteins and dual control of adenylate cyclase. Cell. tivity by isoproterenol. In our opinion, this enhancement of 36:577-581(1984) adenylate cyclase activity by PMA was due to the latter's faciliGrim, W., F. Marks: Effect of tumor promoting phorbol ester on the tation of s-GTP interaction with adenylate cyclase via the normal and isoproterenol elevated level of adenosine 3',5' c monophosphorylation of a protein by protein kinase C. The phosphate in mouse epidermia in vivo. Cancer Res. 34:3128-3134 sequence of events in intact PMN appears to be that PMA acti(1974) vates the soluble protein kinase C, which then binds to the Malski, T. F. P., P. H. Nacchache, M. L. Marsh, J. Kermode, E. L. membrane. As demonstrated earlier, this translocation was esBecker, R. I. Sha'Agi: Pertussis toxin inhibits the rise in the intracellular concentration of free calcium that is induced by chemisential in order to potentialize the effect of isoproterenol on otactic factors in rabbit neutrophils: possible role of the "G proadenylate cyclase, since the effect of PMA was not evident on tein" in calcium mobilization. Biochem. Biophys. Res. Commun. crude membrane. However, when, as here, its effect was tested 124:644-650(1984) on the membrane - plus supernatant mixture, the Ac stimulaNabika, T., Y. Nara, Y. Yamori, W. Lovenberg, J. Endo: Angiotensin tion by isoproterenol was enhanced, indicating previous actiII and phorbol ester enhance isoproterenol and vasoactive intestivation of the soluble PKc in the supernatant. In platelets, pronal peptide (VIP)-induced cyclic AMP accumulation in vascular tein kinase C was previously found to interfere with Ni-medismooth nuclei cells. Biochem. Biophys. Res. Commun. 131:30-55 (1985) ated inhibition of adenylate cyclase (Watanabe, Horn, Bauer and Jakobs 1985). Treatment of intact platelets with TPA also Novogrosky, A., M. Patya, A. L. Rubin, K. H. Stenzel: Inhibition of (Sadrenergic stimulation of lymphocyte adenylate cyclase by phorgreatly affected adenylate cyclase regulation, because it inbol myristate is mediated by activated macrophages. Biochem. Bihibited the guanine nucleotide site. It was suggested that this ophys. Res. Commun. 104:389-393 (1982) inhibition by TPA was due to stimulation of the Ca 2+ -acti- Rebut-Bonneton, C, J. Demignon, P. Sourbier: Joint meeting of the vated phospholipid-dependent protein kinase C. Phorbol Societe Francaise d'Immunologie and British Society for Imester treatment was also reported to desensitize and enhance munology, Spring Meeting-Paris, The Chamelson Press limited, the beta adrenergic responses in smooth muscle cells (Nabika, London(1988),P0479 Nara, Yamori, Lovenberg and Endo 1985) and lymphoma cells Rochette-Egly, C, M. Castagna:k tumor producing phorbol ester inhibits the cyclic AMP response of rat embryo fibroblasts to cate{Bell, Buxton and Burton 1985), and to mediate the inhibition cholamines and prostaglandin Ei (PGEi). FEBS Letter 103: 38of adenylate cyclase by IL2 in human lymphocytes (Beckner 42(1979) and Farrar 1987). With the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP), it has been reported that it does not stimulate adenylate cyclase activity in membrane preparations from leukocytes but can increase cAMP levels in intact cells (Malski, Naccache, Marsh, Kermode,
Solomon, Y., C. Londos, M. Rodbell: A highly sensitive adenylate cyclase assay. Analytical Biochem. 58:541-548 (1974) Tecoma, E., H. Matulsky, A. Traynor, G. Omann, H. Muller, L. A. Sklar: Transient catecholamine modulation of neutrophil activation: kinetic and intracellular aspects of isoproterenol action. J. Leukocyte Biol. 40:629-644 (1986)
Downloaded by: Boston University. Copyrighted material.
Discussion
Horm. metabol. Res. 22 (1990)
C. Rebut-Bonneton, J. Demignon and P. Sourbier
Verghese, M. W., C. D. Smith, R. Snyderman: Potential role of guanine Requests for reprints should be addressed to: nucleotide regulatory protein in chemoattractant-receptor mediated phosphoinositides metabolism, Ca + + metabolization and C. Rebut-Bonneton cellular responses by leukocytes. Biochem. Biophys. Res. ComU262INSERM mun. 127:450-^57(1985) 123 boulevard de Port-Royal Watanabe, Y., F. Horn, S. Bauer, K. H. Jakobs: Protein kinase C interF-75674 Paris Cedex 14 (France) feres with Ni mediated inhibition of human platelet adenylate cyclase. FEBS. Letters 192:23-27 (1985) Wolfson, M., L. C. McPhail, V. W. Nasrallah, R. Snyderman.Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: Potential role in the activation of the respiratory burst enzyme. J. Immunol. 135:2057-2061(1985)
Downloaded by: Boston University. Copyrighted material.
178
Summary The putative role of protein phosphorylation in modulating adenylate cyclase activity in polymorphonuclear neutrophil membranes was assessed using phorbol myristate acetate (PMA) to stimulate the activity of protein kinase C. PMA was demonstrated to enhance the adenylate cyclase activity stimulated by isoproterenol. Key words Adenylate Cyclase - Polymorphonuclear Leukocytes - Isoproterenol - Protein Kinase C - Formyl Methionyl - Leucyl- Phenylalanine
To determine whether PKc changes the response of polymorphonuclear leukocytes (PMN) to isoproterenol, intact PMN, 40000 g-crude membranes or the membranes plus supernatant were treated with phorbol myristate acetate (PMA) before incubation with isoproterenol. PMA was previously shown to induce PKc translocation from the cytosol to the membrane (Wolfson, McPhail, Nasrallah and Snyderman 1985) where PKc was assumed to phosphorylate subunits of the adenylate cyclase system. Adenylate cyclase activity was measured by the rate of ATP conversion into cAMP, using a radioimmunoassay. Materials and Methods Material Phorbol myristate acetate (PMA), N formylmethionyl-leucyl-phenylalanine (FMLP), isoproterenol, isobutylmethylxanthine (IBMX), ATP, GTP, creatine phosphokinase and phosphocreatine were obtained from Sigma. The radioimmunoassay kit for cAMP determination by acetylation was purchased from New England Nuclear.
Introduction Cyclic adenosine monophosphate (cAMP) has long been recognized as the second messenger in many hormonal actions as well as in the control of cell proliferation. The molecular mechanism regulating the enzyme responsible for cAMP synthesis, i. e. adenylate cyclase (Ac), is now well characterized (Gilman 1984) and is known to involve a complex coupling system with different stimulatory or inhibitory receptors, and corresponding GTP binding proteins. The intracellular concentration of cAMP is also regulated by phosphodiesterase. Recent reports suggest that protein kinase C (PKc) is able to modify a component of adenylate cyclase, thus inducing changes in cell cAMP content. Fibroblasts (RochetteEgly and Castagna 1979) and epidermal cells (Grim and Marks 1974) exposed to the phorbol ester tumor promotor 12-0-tetradecanoyl phorbol-13-acetate (TPA) exhibited reduced ability to accumulate cAMP in response to hormones. This phorbol ester also inhibited isoproterenol-stimulated adenylate cyclase in human peripheral blood mononuclear cells (Novogrosky Patya, Rubin and Stenzel 1982).
Horm. metabol. Res. 22 (1990) 175-178 © Georg Thieme Verlag Stuttgart • New York
Adenylate cyclase assays in preparations
membranous
Venous blood was obtained on heparin (10 U/ml) from healthy adult donors. Purified PMN preparations were isolated from it, using Ficoll/Hypaque gradients (Mono-poly resolving medium, Flow Laboratories) and erythrocytes were lysed in hypotonic medium. Cells were suspended in an ice-cold buffered solution pH 7.4 (KRP) containing, in mM, 138 NaCl, 2.7 KC1, 8.1 Na2HPO4, 1.5 K2HPO4 and 1.0 MgCl2. Intact PMN were incubated at 37 °C for the indicated time in KRP solution containing 0.01 mM Ca 2+ (20 • 106 cells/ml) in the presence or absence of PMA. They were then quickly transferred to an ice bath and centrifuged for 8 min at 4 °C and 600 g. The pellet was diluted to reach 40 • 10 cells perml in bufferA(pH7.7) containing in mM, 50 Tris HC1, 10 MgCl2 and 2 EDTA. The suspension was homogenized three times for 30 sec each with a Potter homogenizer and centrifuged for 8 min at 200 g to remove nuclei and large debris. The resulting supernatant was centrifuged at 41000 g for 12 min and the remaining pellet containing the cell membranes and subcellular particles was resuspended by 10 sec sonication in 500 |xl of buffer B (pH 7.5) containing 75 mM Tris HC1,25 mMMgCl2and 10-4 M IBMX, and stored on ice. The protein concentration in the pellet
Received: 6 March 1989
Accepted: 31 July 1989
Downloaded by: Boston University. Copyrighted material.
1
Horm. metabol. Res. 22 (1990)
C. Rebut-Bonneton, J. Demignon and P. Sourbier
was adjusted to 6 ug/20 ul by dilution in buffer B, and the protein concentration in the supernatant was adjusted to 2 ug/20 ul. A reaction mixture was prepared on ice by sequentially adding 20 ul of membrane preparation, and/or 10 ul of supernatant and 10 ul of stimulant. The preliminary incubation with PMA was performed for 5 min at 0 °C; afterwards, isoproterenol was added to the final mixture containing 0.1 mM ATP, 0.1 mM GTP, 5 mM phosphocreatine and 0.1 mg/ml of creatine phosphokinase (phosphocreatine and creatine phosphokinase were used to generate ATP). Ac activation was initiated by placing the tubes in a 37 C water bath for 10 min and was stopped by icing the tubes. Accumulated cAMP was measured by radioimmunoassay using a NEN kit, and not by the usual procedure designed to evaluate the production of labelled cAMP from the 32P-ATP substrate (Solomon, Londos and Rodbell 1974) 200 ul of ice-cold acetate buffer and 5 ul of acetylation reagent, prepared by mixing 100 ul of triethylamine and 50 ul of acetic anhydride in a glass tube, were added. Three minutes were allowed for
acetylation at room temperature, and 100 ul of each sample was then transferred into plastic assay tubes. 100 ulof ,25I-cAMPand 100 ulof antiserum complex were then added to these tubes as indicated in the kit for cAMP determination. Results As shown by the data in Table 1,10 - 4 or 10~5 M isoproterenol significantly stimulated adenylate cyclase activity of membrane preparations, thus raising cAMP levels.
Table 1 Effect on PMN adenylate cyclase activity of pretreating PMN membranes with 10 ng/ml PMA for 10 min before their stimulation by isoproterenol. The reaction mixture contained 0.1 mM ATP, 0.1 mM GTP, 5 mM creatine phsophate, and 0.1 mg/ml creatine phosphokinase. cAMP accumulation was measured by radioimmunoassay. Control
100 ± 1 4 (3)
Isoproterenol 10- 5 M
237 ± 4 9 * * * (3)
Isoproterenol 10- 4 M
236 ± 19*** (3)
10 ng/ml PMA + 10-5 M isoproterenol
250 ± 2 2 * * * (3)
100ng/mlPMA + 10 M isoproterenol
207 ± 2 9 * * * (3)
Fig. 1 Effect of pretreatment of the supernatant + crude membrane preparation on adenylate cyclase activation by isoproterenol. cAMP accumulation was measured by radioimmunoassay. The "generator" of ATP was a mixture containing 0.1 mM ATP, 5 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase and 0.1 mM GTP. 1: membrane + supernatant 2: membrane + supernatant + 10~5 M isoproterenol 3,4: membrane + supernatant + PMA (10and 10 ng/ml respectively) + 10 _5 M isoproterenol. The mean control value was taken as 100 and the other values were calculated on this basis. Results are means ± SD. a P < 0.01 in comparison with control value b P < 0.01 in comparison with isoproterenol alone (variance analysis)
The mean control value was taken as 100 and the other values were calculated on this basis. Number of experiments is in parenthesis. ***P < 0.01 (variance analysis) versus to the control.
Table 2 Effect on the adenylate cyclase activity in PMN membranes of treating intact PMN with PMA for 5 min at 37 °C before incubating the membranes for 10 min at 37 °C with 10~5 M isoproterenol. PMN membranes from intact PMN treated with PMA for 5 min (ng/ml)
Control PMN membranes
Control
Isoproterenol 10-5M
8.99 ± 1.60 (3)
*** 15.40 ±1.83 (3)
0.1
1
10
11.90 ±3.64 (3)
10.31 ± 1.15 (6)
9.94 ±1.15 (6)
*** 18.90 ±1.83 (3)
***
22.07 ± 4.68ooc) (6)
* * +
26.84 ± 5.21 °°° (6)
cAMP accumulation in the presence of a reaction mixture containing 0.1 mM ATP, 0.1 mM GTP, 5 mM creatine phosphate and 0.1 mg/ml creatine phosphokinase. cAMP was measured by radioimmunoassay. Results are expressed in fmoles/min • |ig membrane protein (means ± SD). * * *P < 0.01 versus to the corresponding control """variance analysis of pretreated versus control PMN () number of experiments is in parenthesis
Downloaded by: Boston University. Copyrighted material.
176
Adenylate Cyclase in PMN
Horm. metabol. Res. 22 (1990)
Pretreatment of plasma membranes for 10 min with 10 or 100 ng/ml PMA did not enhance the degree of adenylate cyclase activation by these isoproterenol concentrations. The supernatant alone did not show any detectable Ac activity, and the addition of supernatant to the membranous preparation did not alter the response of Ac to isoproterenol; pretreatment of the membrane + supernatant mixture with PMA for 5 min at 0 °C before adding isoproterenol for Ac activation enhanced this activation (Fig. 1).
Becker and Sha'agi 1984). Consequently, this agent probably exerts its effect through the production of intermediate agents and/or requires substances available in intact cells but not in membrane preparations. Here, PKc should have been the intermediate agent but the pretreatment of intact PMN by PMA did not permit subsequent incubation with FMLP to raise Ac activity (Rebut-Bonneton, Demignon and Sourbier1988).However according to Verghese, Smith and Snyderman (1988), the chemoattractant might be coupled to an alternative effector mechanism via an N protein.
Pretreatment of intact PMN for 5 min at 37 °C with PMA also enhanced the Ac activation by isoproterenol in plasma membranes (Table 2). After incubation with isoproterenol a positive correlation was found between the concentration of PMA in the incubation medium and the adenylate cyclase activity in the membrane (r = 0.702, P < 0.01). PMA alone did not exert any detectable stimulation.
In conclusion, in PMN, PMA was shown to be an agent responsible for modulating the cAMP level through the PKc stimulation. In some cases, the effect of PMA could help in discriminating between a cell content cAMP increase due to a direct action on the adenylate cyclase system or to another mechanism. Acknowledgements This work was carried out in U 294 INSERM, 46 rue
The fact that in these membrane preparations Huchard, 750 18 Paris. ATP was converted into cAMP through hormone stimulation, References and also through the non specific agent NaF (result not shown) demonstrates that the catalytic unit of Ac remained inBeckner, S. K., W. L. Farrar: Inhibition of adenylate cyclase by IL2 in tact and functional. The presence of guanine nucleotides was human T lymphocytes is mediated by protein kinase C. Biochem. an absolute requirement for Ac activation by isoproterenol but Biophys. Res. Commun. 145:176-181 (1987) was not necessary for its activation by NaF stimulation. Iso- Bell, J. D., I. Buxton, L. L. Burton: Enhancement of adenylate cyclase proterenol was previously shown to activate Ac (Tecoma, activity in S49 lymphoma cells by phorbol ester: putative effect of Matulsky, Traynor, Omann, Mutter and Sklar 1986). Here, C kinase on Ls-GTP catalytic subunit interaction. J. Biol. Chem. PMA was demonstrated to reinforce the stimulation of Ac ac280:2625-2628(1985) Gilman, A. G.: G proteins and dual control of adenylate cyclase. Cell. tivity by isoproterenol. In our opinion, this enhancement of 36:577-581(1984) adenylate cyclase activity by PMA was due to the latter's faciliGrim, W., F. Marks: Effect of tumor promoting phorbol ester on the tation of s-GTP interaction with adenylate cyclase via the normal and isoproterenol elevated level of adenosine 3',5' c monophosphorylation of a protein by protein kinase C. The phosphate in mouse epidermia in vivo. Cancer Res. 34:3128-3134 sequence of events in intact PMN appears to be that PMA acti(1974) vates the soluble protein kinase C, which then binds to the Malski, T. F. P., P. H. Nacchache, M. L. Marsh, J. Kermode, E. L. membrane. As demonstrated earlier, this translocation was esBecker, R. I. Sha'Agi: Pertussis toxin inhibits the rise in the intracellular concentration of free calcium that is induced by chemisential in order to potentialize the effect of isoproterenol on otactic factors in rabbit neutrophils: possible role of the "G proadenylate cyclase, since the effect of PMA was not evident on tein" in calcium mobilization. Biochem. Biophys. Res. Commun. crude membrane. However, when, as here, its effect was tested 124:644-650(1984) on the membrane - plus supernatant mixture, the Ac stimulaNabika, T., Y. Nara, Y. Yamori, W. Lovenberg, J. Endo: Angiotensin tion by isoproterenol was enhanced, indicating previous actiII and phorbol ester enhance isoproterenol and vasoactive intestivation of the soluble PKc in the supernatant. In platelets, pronal peptide (VIP)-induced cyclic AMP accumulation in vascular tein kinase C was previously found to interfere with Ni-medismooth nuclei cells. Biochem. Biophys. Res. Commun. 131:30-55 (1985) ated inhibition of adenylate cyclase (Watanabe, Horn, Bauer and Jakobs 1985). Treatment of intact platelets with TPA also Novogrosky, A., M. Patya, A. L. Rubin, K. H. Stenzel: Inhibition of (Sadrenergic stimulation of lymphocyte adenylate cyclase by phorgreatly affected adenylate cyclase regulation, because it inbol myristate is mediated by activated macrophages. Biochem. Bihibited the guanine nucleotide site. It was suggested that this ophys. Res. Commun. 104:389-393 (1982) inhibition by TPA was due to stimulation of the Ca 2+ -acti- Rebut-Bonneton, C, J. Demignon, P. Sourbier: Joint meeting of the vated phospholipid-dependent protein kinase C. Phorbol Societe Francaise d'Immunologie and British Society for Imester treatment was also reported to desensitize and enhance munology, Spring Meeting-Paris, The Chamelson Press limited, the beta adrenergic responses in smooth muscle cells (Nabika, London(1988),P0479 Nara, Yamori, Lovenberg and Endo 1985) and lymphoma cells Rochette-Egly, C, M. Castagna:k tumor producing phorbol ester inhibits the cyclic AMP response of rat embryo fibroblasts to cate{Bell, Buxton and Burton 1985), and to mediate the inhibition cholamines and prostaglandin Ei (PGEi). FEBS Letter 103: 38of adenylate cyclase by IL2 in human lymphocytes (Beckner 42(1979) and Farrar 1987). With the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP), it has been reported that it does not stimulate adenylate cyclase activity in membrane preparations from leukocytes but can increase cAMP levels in intact cells (Malski, Naccache, Marsh, Kermode,
Solomon, Y., C. Londos, M. Rodbell: A highly sensitive adenylate cyclase assay. Analytical Biochem. 58:541-548 (1974) Tecoma, E., H. Matulsky, A. Traynor, G. Omann, H. Muller, L. A. Sklar: Transient catecholamine modulation of neutrophil activation: kinetic and intracellular aspects of isoproterenol action. J. Leukocyte Biol. 40:629-644 (1986)
Downloaded by: Boston University. Copyrighted material.
Discussion
Horm. metabol. Res. 22 (1990)
C. Rebut-Bonneton, J. Demignon and P. Sourbier
Verghese, M. W., C. D. Smith, R. Snyderman: Potential role of guanine Requests for reprints should be addressed to: nucleotide regulatory protein in chemoattractant-receptor mediated phosphoinositides metabolism, Ca + + metabolization and C. Rebut-Bonneton cellular responses by leukocytes. Biochem. Biophys. Res. ComU262INSERM mun. 127:450-^57(1985) 123 boulevard de Port-Royal Watanabe, Y., F. Horn, S. Bauer, K. H. Jakobs: Protein kinase C interF-75674 Paris Cedex 14 (France) feres with Ni mediated inhibition of human platelet adenylate cyclase. FEBS. Letters 192:23-27 (1985) Wolfson, M., L. C. McPhail, V. W. Nasrallah, R. Snyderman.Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: Potential role in the activation of the respiratory burst enzyme. J. Immunol. 135:2057-2061(1985)
Downloaded by: Boston University. Copyrighted material.
178
