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Inhibition of adenylate cyclase activity by polyamines in human erythrocyte plasma membranes
Life Sciences, Vol. 46, pp. 43-47 Printed in the U.S.A.
Pergamon Press
INHIBITION OF ADENYLATE CYCLASE ACTW1TY BY POLYAMINES IN HUMAN ERYTHROCYTE PLASMA MEMBRANES Naim A. Khan, V. Quemener and J. - Ph. Moulinoux Department of Cell Biology, Central Hospital of University (C.H.U.) of Rennes, 2 Av du Pr. IMon Bernard, 35043 Rennes Cedex (France). (Received in final form November 6, 1989)
Summary Polyamines (spermidine, spermine and putrescine) inhibited the adenylate cyclase activity in a concentration dependent manner in human erythrocyte plasma membranes. Spermidine (Spd) exhibited more inhibitory effect than spermine (Spm) and putrescine (Put). On the contrary, the addition of amino acids (arginine, glutamine and lysine) did not influence the basal enzyme activity. Other cations (polylysine, polyarginine and polyglutamine) also did not affect the enzyme activity. Addition of all the three polyamines (Spd, Spm and Put) in the reaction mixture exhibited moderate inhibitory effect on the adenylate cyclase activity whether it was basal or activated with sodium fluoride or with forskolin. Since the three polyamines exhibited maximum inhibitory effect at 10 ktM concentration which is within physiological limit for mammalian tissues, we suggest that there may be a regulatory function of these molecules on adenylate cyclase activity in human erythrocytes. Introduction A number of protein kinases have been identified in red blood cells. In human erythrocytes, cyclic AMP - dependent and independent protein kinases are reported to be inherent components of the cell membranes (1, 2). Although the role of these kinases in membrane functions remains to be established, it is known that the red cell membranes contai~ several proteins which are phosphorylated by these kinases and the enzymes use ATP as the phosphoryl donors (3). Erythrocytes from chicken (4), frog (5), turkey (6) and pigeon (7, 8) have been reported to be desensitized with catecholamines because these cells possess 13- adrenergic receptors suggesting the process of desensitization may be mediated via adenylate cyclase system. On the other hand, the capacity of polyamines and other polycationic peptides to stimulate casein kinase 2 has been reported (9). Moreover, there is lack of information on the direct studies on adenylate cyclase system in mammalian red blood cells except a recent study on rat erythrocyte plasma membranes (10). There are several reports on the influence of polyamines on adenylate cyclase activity in nucleated cells (11- 14) but no study has yet been carried out on the effect of polyamines on this enzyme in anuclear human red blood cells. We also used basic amino acids and other polycations to further assess the specificity of the effects of polyamines on adenylate cyclase activity. Materials and Methods Fresh human blood was procured from Centre Regional de Transfusion Sanguine de Rennes (France) and washed three times with PBS (phosphate buffered saline, pH 7.4). The erythrocyte plasma membranes were prepared as described elsewhere (15). The reaction mixture (1 ml) for adenylate cyclase activity contained 100 I.tl of the plasma membranes (1 mg of protein / ml), 50 mM Tris-HCl, pH 7.4, 10 mM KC1, 5 m M MgCI2, 10 mM creatine phosphate, 15 I.tg/ ml creatine kinase, 3 mM dithiothreitol, 0.1 mM 3H-ATP and increasing concentrations of one of the polyamines (Spd, Spm or Put) and amino acids (asparagine, glutamine and lysine) or other cations (polylysine, polyglutamine an~polyarginine) or fixed concentrations of sodium fluoride or forskolin or no addition (basal). ~H-ATP was purchased from Amersham Radiochemicals, France. The reaction was started by the addition of the plasma membranes and terminated by adding 100 ~1 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
44
Adenylate Cyclase in Human Erythrocytes
Vol. 46, No. i, 1990
of 2 % sodium dodesylsulphate, 40 mM ATP and 1.4 mM cAMP at pH 7.5. The adenylate cyclase activity was measured according to Salomon et al. (16). Protein was determined according to Lowry et al. (17). Results and Discussion Figure 1 shows that all the three polyamines i.e. Spd, Spm and Put inhibited the basal adenylate cyclase activity in human erythrocytes. Spd exhibited more inhibitory effect than Spm and Put. Arginine (Arg), glutamine (Gin) and lysine (Lys) did not appreciably influence the enzyme activity whatever the concentration of the amino acids was used. Table I further confirms the inhibitory effects of polyamines on adenylate cyclase activity in human erythrocyte plasma membranes. The polyamines when used at 10 ~tM concentrations exhibited maximum inhibitory effect while the three polyamines altogether (Spd, Spm and Put), in a reaction mixture containing each 2.5 lxM, exhibited moderate inhibitory effects. Polycations (polylysine, polyglutamine and polyarginine) did not affect the the enzyme activity at 10 ~tM concentrations.
5
Z)
1
0
10 20 30 40 Polyamines / Amino acids added, ktM
Figure 1. Effects of different concentrations of polyamines and amino acids on the basal activity of adenylate cyclase in human erythrocytes. Erythrocyte plasma membranes were isolated and incubated at increasing concentrations of spermidine (A), spermine (O) and putrescine (It), arginine (m), lysine (A) and glutamine (<~). The cAMP formed was measured as described in the Materials and Methods. All the assay samples were in quadruplicate. Forskolin, a diterpene from the root of Coleus f o r s k o l i i , is known to directly activate the catalytic unit of the adenylate cyclase system (17) and this activation may be mediated through a low affinity binding sites for forskolin (19). In addition, the activation of this enzyme system by forskolin requires guanine nucleotides stimulatory and inhibitory (Gs) proteins (20). In this case the number of high affinity binding sites for froskolin are increased (21). The effect of forskolin depend on the formation of a complex between Gs and catalytic unit of the enzyme (9). The inhibitory effect of polyamines on the froskolin activated adenylate cyclase activity may be postulated as a destabilizing effect of polyamines on the interaction between Gs and catalytic unit of the enzyme. Polyamines have been recently reported to bind with catalytic unit of the protein kinase C in rat brain (22) but the mechanism of interaction has not been well characterized. Nevertheless, this study suggests that polyamines might have an influence on the function of Gs protein in human
Vol. 46, No. i, 1990
Adenylate Cyclase in Human Erythrocytes
45
erythrocytes. In this study, polyamines also inhibited the basal and sodium fluoride activated enzyme activity and this is in agreement with the findings of several investigators who have reported that polyamines inhibit the basal adenylate cyclase activity in a number of nucleated cells (11 - 14) though there is also a contradictory report on the several fold increase of the enzyme activity by polyamines in human spermatozoites (23). It seems clear that the effect of polyamines on adenylate cyclase system is not merely a charged interaction between cation and negatively charged membrane; if it were so, the positive charged amino acids (Arg and Lys) or polycations (polylysine and polyarginine) would have a similar or modified effects. However, it is most probable that charged interactions may play a role as polyamines have been shown to alter the lateral mobility of erythrocyte membrane proteins and to stabilize the erythrocyte structure by binding with cytoskeleton proteins (24, 25). In almost all in vitro biological systems Spm is the most potent activator or inhibitor of biochemical steps and this has been attributed to the higher cationic charges of Spm than Stxt (26). This always calls into question whether or not we are dealing with something pharmacological. In addition, Spm has been found to exert higher inhibitory effects than Spd on nuclear adenylate cyclase from Physarum polycephalum while the activity of cytoplasmic adenylate cyclase was not affected by either of polyamines (27).The present study demonstrates that Spd in equirnolar concentrations is more potent inhibitor of adenylate cyclase activity than Spm.Glutamine, a neutral amino acid, and polyglutamine also did not influence the enzyme activity. In another study, we have recently observed that the negative charged moities of the cell membranes are not true polyamine interaction sites and rather they prevent a large quantity of polyamines to bind with the cellular glycoproteins in murine leukemia cells (28). Moreover, human erythrocytes also possess a Na + - dependent polyamine transport system which is neither shared nor influenced by amino acids (29). Table - I Effects of polyamines on basal, NaF and Forskolin activated adenylate cyclase activity in human erythrocyte plasma membranes. Addition
Adenylate cyclase activity (pmol / min / mg protein)
Basal (None) Spd Spm Put Polyamines*** Polylysine Polyglutamine Polyarginine NaF NaF+ Put NaF+ Spm Naf + Spd Naf + Polyamines*** Forskolin Forskolin + Put Forskolin + Spm Forskolin + Spd Forskolin + Polyamines***
4.0 + 0.4* 1.1 + 0.0" 2.0 + 0.1" 3.0 + 0.3** 2.3 + 0.1" 3.2 + 0.4§ 3.5 + 0.3§ 3.8 + 0.3§ 7.5 + 0.7* 6.5 + 0.2* 5.2 + 0.3* 3.2 + 0.5* 5.0 + 0.9* 15.2 + 1.7 * 13.1 + 1.3" 10.4 + 1.6" 6.3 + 1.3" 10.5 + 1.6"
Human erythrocyte plasma membranes were added with 10 ktM of either of the additions i.e. Spd, Spin, NaF or Forskolin. Data are mean + SD of quadruplicate assay samples. ***The group received 2.5 ~tM of each of all the three Polyamines (Spd, Spm and Put.). Each of the polycations was also used at 10 IxM concentration. Data differ significantly in comparison with control values following to Mann Whitney test of significance *(p < 0.001) ; **(p < 0.01). §Insignificant data in comparison with basal values.
46
Adenylate Cyclase in Human Erythrocytes
Vol. 46, No. i, 1990
The regulatory implications of the observed polyamine induced attenuation of the adenylate cyclase activity in the present study in human erythrocyte plasma membranes remains obscure. However, it might be argued that polyamines might act for self regulatory function. Their systhesis during erythroid maturation of red blood ceils coupled to adenylate cyclase induction of ornithine decarboxylase (ODC), a rate limiting enzyme of polyamine systhesis via the phosphorylation by cAMP dependent kinase would require cAMP for sustained activation. Such a state of affairs necessary to maintain the steady state levels of polyamines has been shown by Caldarera et al (14). They further reported that treatment of primary heart cells with DFMO (alfa difluoromethylornithine), an irreversible inhibitor of ODC activity, resulted in low intracellular polyamine concentrations and high cAMP levels and adenylate cyclase activity (12). It is possible that 10 p.M concentrations of PA may not ordinarily be found in the free state of intact ceils (30), nevertheless, we may propose that the polyamines might play a regulatory role, by altering the activity of second messengers (adenylate cyclase), on the cell surface though the role of polyamines as second messengers has also been suggested by several investigators (31, 32). This idea is further supported by the recent findings of Fan and Koenig (33) who have observed that depletion of intracellular polyamines in heart cells abrogated the stimulation of Ca++ influx, hexose and amino acid transport evoked by isoproterenol. Moreover, polyamines may enhance the phosphorylation of cell proteins following of activation of polyamine dependent protein kinases (34, 35). Further studies are in progress to investigate whether polyamines affect the homologous or heterogolous desensitization of B - adrenergic receptors in chicken erythrocytes and with other cells. It will, in addition, be determined what influence PA have on the generation of cAMP by stimulating Ca++ influx. Acknowledgements This work was supported by research grants from CNRS and a Post Doc Fellowship to one of the authors (NAK) from Association pour Recherche Contre le Cancer (France). 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
References C.E. GUTHROW, J.E. ALLEN and H. RASMUSSEN, J. Biol. Chem. 247, 8145 - 8153 (1972). M.M. HOSEY and M. TAO, Biochemistry. 15, 1561 - 1568 (1976). K.W. SIMKOWSKY and M.TAO, J. Biol. Chem. 255, 6456 - 6461 (1980). P.J.RAVAL and D. ALLEN, Biochem. J. 231, 179 - 183 (1985). J.M. STADEL, B. STRULOVICI, P. NAMBI, T.N. LAVIN, M.M. BRIGGS, M.G. CARON and R.J. LEFKOWITZ, J. Biol. Chem. 258, 3032 - 3038 (1983). D.R. SIBLEY, J.R. PETERS, P. NAMBI, M.G. CARON and R.J. LEFKOWITZ, J. Biol. Chem. 259, 9742 - 9749 (1984). J.M. STADEL, R. REBAR and S.T. CROOKE, Biochem. J. 252, 771 - 776 (1988). K.M. POPOV, T.V. BULARGINA and E.S. SEVERIN, Biokhemiya (Russian).50, 443 446 (1985). L. LEVIA, D. CARRASCO, A. TAYLOR, M. VELIZ, C. GONZALEZ, C.C. ALLENDE and J.E. ALLENDE, Biochem. Int.14, 77-717 (1987). O. TAKEDA, K. YAMASAKI, H. KOHDA, A. YAMASHITA, T. KURIKAWA and S. ISHIBASHI, J. Pharmacobio. - Dyn. 11,377 -380 (1988). C. CLO, C.M. CARDARERA, B. TANTINI, D. BENALAL and U. BACHRACH, Biochem. J. 182, 641 - 649 (1979). C. CLO, C. PIGANATTI, S. MANFRONI, S. MARMIROLI and B. TANTINI, in Biochemical Studies of Natural Polyamines. (eds. C.M. Calderara, C. Clo and C. Guarnieri). CLUEB Bologna, Italy. 117 - 125 (1986). C. CLO, B. TANTINI, C. PIGANATTI, S. MARMIROLI and C.M. CALDARERA, in Persoectives in Polvamine Research. (eds. A. Perin, G. Scalabrino, A. Sessa and M.E. Ferioli).Wichtig Edit-ore, Milano, Italy. 45 - 48 (1988). C.M. CALDARERA, B. TANTINI, S. MARMIROLI, C. PIGNATTI and C. CLO, in .R...gcent Progress in Polvamine Research. (eds. L. Selmeci, M.E. Brosman and N. Seiler) 109 - 119 (1985). R.S KENT, A.D. LEAN and R.J. LEFKOWITZ, Mol. Pharmacol.17, 14 - 23 (1980).
Vol. 46, No. I, 1990
16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.
Adenylate Cyclase in Human Erythrocytes
47
Y. SALOMON, C. LONDOS and M. RODBELL, Anal. Biochem. 58, 541 - 548 (1974). O.H. L O W R Y , N . J . R O S E B O R O U G H , A.L. F A R R and R.J. R A N D E L L , J. Biol. Chem.19~, 265 - 275 (1951). K.B. SIAMON and J.W. DALY, J. Cyclic Nucleotide Res. 7, 201 - 224 (1981). D.A. GREEN and R.B. CLARCK, J. Cyclic Nuleotide Res.8, 337 - 346 (1981). P.A. INSEL, D. STANGEL, N. FERRY and J. HANOVNE, J. Biol. Chem. 257, 7485 7490 (1985). G. BAII~AGLIA, A.B. NORAMN, E.J. HESS and I. CREESE, J. Neurochem. 46, 1180 1185 (1986). G. MEZZETI, G. M. MONTI and M.S. MORUZZI, Life Sci. 42, 2293 - 2298 (1988). G.V. SHAH, A.R. SETH and S.S. RAO, Experientia, 31,631 - 632 (1974). M. SCHINDLER, D. KOPPEL and M.P. SCHEETZ, Proc. Natl. Acad. Sci., 77, 1457 1461 (1980). J.W. WYSE and D.A. BUTTERFIELD. Biochem. Biophys. Acta. 941,141 - 149 (1988). F. SCHUBER, Biochem. J. 260, 1- 10 (1989). V.J. ATMAR, J.A. WESTLAND, G. G A R C I A and G.D. KUEHN, Biochem. Biophys. Res. Commun. 68,561 - 569 (1976). N.A. KHAN, V. QUEMENER and J.Ph. MOULINOUX, Cell Mol.Biol. 3 5 , 2 1 5 - 224 (1989). N.A. KHAN, V. QUEMENER and J.Ph. MOULINOUX, Biochem. A r c h 5 , 161 - 169 (1989). D.M. OTA, K. NISHIOKA, M. FOULKES and V.B. GROSSIE, J. Clin. Oncol.2, 1157 1164 (1984). M. S H R A M M and M. SELIGNER, Science. 225, 1350 - 1356 (1984). Y. NISHIZUKA, Science. 225, 1365 - 1370 (1984). C.C. F A N and H. KOENIG, J. Mol. Cell Cardiol. 20, 789 - 799 (1988). C. COCHET and E.M. CHAMBAZ, Mol. Cell Endocrinol. 30, 247 - 266 (1983). Z.B. LEVASHOVA and T.M. MOROZOVA, Biokhemiya (Russian). 53, 251 - 255 (1988).
Pergamon Press
INHIBITION OF ADENYLATE CYCLASE ACTW1TY BY POLYAMINES IN HUMAN ERYTHROCYTE PLASMA MEMBRANES Naim A. Khan, V. Quemener and J. - Ph. Moulinoux Department of Cell Biology, Central Hospital of University (C.H.U.) of Rennes, 2 Av du Pr. IMon Bernard, 35043 Rennes Cedex (France). (Received in final form November 6, 1989)
Summary Polyamines (spermidine, spermine and putrescine) inhibited the adenylate cyclase activity in a concentration dependent manner in human erythrocyte plasma membranes. Spermidine (Spd) exhibited more inhibitory effect than spermine (Spm) and putrescine (Put). On the contrary, the addition of amino acids (arginine, glutamine and lysine) did not influence the basal enzyme activity. Other cations (polylysine, polyarginine and polyglutamine) also did not affect the enzyme activity. Addition of all the three polyamines (Spd, Spm and Put) in the reaction mixture exhibited moderate inhibitory effect on the adenylate cyclase activity whether it was basal or activated with sodium fluoride or with forskolin. Since the three polyamines exhibited maximum inhibitory effect at 10 ktM concentration which is within physiological limit for mammalian tissues, we suggest that there may be a regulatory function of these molecules on adenylate cyclase activity in human erythrocytes. Introduction A number of protein kinases have been identified in red blood cells. In human erythrocytes, cyclic AMP - dependent and independent protein kinases are reported to be inherent components of the cell membranes (1, 2). Although the role of these kinases in membrane functions remains to be established, it is known that the red cell membranes contai~ several proteins which are phosphorylated by these kinases and the enzymes use ATP as the phosphoryl donors (3). Erythrocytes from chicken (4), frog (5), turkey (6) and pigeon (7, 8) have been reported to be desensitized with catecholamines because these cells possess 13- adrenergic receptors suggesting the process of desensitization may be mediated via adenylate cyclase system. On the other hand, the capacity of polyamines and other polycationic peptides to stimulate casein kinase 2 has been reported (9). Moreover, there is lack of information on the direct studies on adenylate cyclase system in mammalian red blood cells except a recent study on rat erythrocyte plasma membranes (10). There are several reports on the influence of polyamines on adenylate cyclase activity in nucleated cells (11- 14) but no study has yet been carried out on the effect of polyamines on this enzyme in anuclear human red blood cells. We also used basic amino acids and other polycations to further assess the specificity of the effects of polyamines on adenylate cyclase activity. Materials and Methods Fresh human blood was procured from Centre Regional de Transfusion Sanguine de Rennes (France) and washed three times with PBS (phosphate buffered saline, pH 7.4). The erythrocyte plasma membranes were prepared as described elsewhere (15). The reaction mixture (1 ml) for adenylate cyclase activity contained 100 I.tl of the plasma membranes (1 mg of protein / ml), 50 mM Tris-HCl, pH 7.4, 10 mM KC1, 5 m M MgCI2, 10 mM creatine phosphate, 15 I.tg/ ml creatine kinase, 3 mM dithiothreitol, 0.1 mM 3H-ATP and increasing concentrations of one of the polyamines (Spd, Spm or Put) and amino acids (asparagine, glutamine and lysine) or other cations (polylysine, polyglutamine an~polyarginine) or fixed concentrations of sodium fluoride or forskolin or no addition (basal). ~H-ATP was purchased from Amersham Radiochemicals, France. The reaction was started by the addition of the plasma membranes and terminated by adding 100 ~1 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
44
Adenylate Cyclase in Human Erythrocytes
Vol. 46, No. i, 1990
of 2 % sodium dodesylsulphate, 40 mM ATP and 1.4 mM cAMP at pH 7.5. The adenylate cyclase activity was measured according to Salomon et al. (16). Protein was determined according to Lowry et al. (17). Results and Discussion Figure 1 shows that all the three polyamines i.e. Spd, Spm and Put inhibited the basal adenylate cyclase activity in human erythrocytes. Spd exhibited more inhibitory effect than Spm and Put. Arginine (Arg), glutamine (Gin) and lysine (Lys) did not appreciably influence the enzyme activity whatever the concentration of the amino acids was used. Table I further confirms the inhibitory effects of polyamines on adenylate cyclase activity in human erythrocyte plasma membranes. The polyamines when used at 10 ~tM concentrations exhibited maximum inhibitory effect while the three polyamines altogether (Spd, Spm and Put), in a reaction mixture containing each 2.5 lxM, exhibited moderate inhibitory effects. Polycations (polylysine, polyglutamine and polyarginine) did not affect the the enzyme activity at 10 ~tM concentrations.
5
Z)
1
0
10 20 30 40 Polyamines / Amino acids added, ktM
Figure 1. Effects of different concentrations of polyamines and amino acids on the basal activity of adenylate cyclase in human erythrocytes. Erythrocyte plasma membranes were isolated and incubated at increasing concentrations of spermidine (A), spermine (O) and putrescine (It), arginine (m), lysine (A) and glutamine (<~). The cAMP formed was measured as described in the Materials and Methods. All the assay samples were in quadruplicate. Forskolin, a diterpene from the root of Coleus f o r s k o l i i , is known to directly activate the catalytic unit of the adenylate cyclase system (17) and this activation may be mediated through a low affinity binding sites for forskolin (19). In addition, the activation of this enzyme system by forskolin requires guanine nucleotides stimulatory and inhibitory (Gs) proteins (20). In this case the number of high affinity binding sites for froskolin are increased (21). The effect of forskolin depend on the formation of a complex between Gs and catalytic unit of the enzyme (9). The inhibitory effect of polyamines on the froskolin activated adenylate cyclase activity may be postulated as a destabilizing effect of polyamines on the interaction between Gs and catalytic unit of the enzyme. Polyamines have been recently reported to bind with catalytic unit of the protein kinase C in rat brain (22) but the mechanism of interaction has not been well characterized. Nevertheless, this study suggests that polyamines might have an influence on the function of Gs protein in human
Vol. 46, No. i, 1990
Adenylate Cyclase in Human Erythrocytes
45
erythrocytes. In this study, polyamines also inhibited the basal and sodium fluoride activated enzyme activity and this is in agreement with the findings of several investigators who have reported that polyamines inhibit the basal adenylate cyclase activity in a number of nucleated cells (11 - 14) though there is also a contradictory report on the several fold increase of the enzyme activity by polyamines in human spermatozoites (23). It seems clear that the effect of polyamines on adenylate cyclase system is not merely a charged interaction between cation and negatively charged membrane; if it were so, the positive charged amino acids (Arg and Lys) or polycations (polylysine and polyarginine) would have a similar or modified effects. However, it is most probable that charged interactions may play a role as polyamines have been shown to alter the lateral mobility of erythrocyte membrane proteins and to stabilize the erythrocyte structure by binding with cytoskeleton proteins (24, 25). In almost all in vitro biological systems Spm is the most potent activator or inhibitor of biochemical steps and this has been attributed to the higher cationic charges of Spm than Stxt (26). This always calls into question whether or not we are dealing with something pharmacological. In addition, Spm has been found to exert higher inhibitory effects than Spd on nuclear adenylate cyclase from Physarum polycephalum while the activity of cytoplasmic adenylate cyclase was not affected by either of polyamines (27).The present study demonstrates that Spd in equirnolar concentrations is more potent inhibitor of adenylate cyclase activity than Spm.Glutamine, a neutral amino acid, and polyglutamine also did not influence the enzyme activity. In another study, we have recently observed that the negative charged moities of the cell membranes are not true polyamine interaction sites and rather they prevent a large quantity of polyamines to bind with the cellular glycoproteins in murine leukemia cells (28). Moreover, human erythrocytes also possess a Na + - dependent polyamine transport system which is neither shared nor influenced by amino acids (29). Table - I Effects of polyamines on basal, NaF and Forskolin activated adenylate cyclase activity in human erythrocyte plasma membranes. Addition
Adenylate cyclase activity (pmol / min / mg protein)
Basal (None) Spd Spm Put Polyamines*** Polylysine Polyglutamine Polyarginine NaF NaF+ Put NaF+ Spm Naf + Spd Naf + Polyamines*** Forskolin Forskolin + Put Forskolin + Spm Forskolin + Spd Forskolin + Polyamines***
4.0 + 0.4* 1.1 + 0.0" 2.0 + 0.1" 3.0 + 0.3** 2.3 + 0.1" 3.2 + 0.4§ 3.5 + 0.3§ 3.8 + 0.3§ 7.5 + 0.7* 6.5 + 0.2* 5.2 + 0.3* 3.2 + 0.5* 5.0 + 0.9* 15.2 + 1.7 * 13.1 + 1.3" 10.4 + 1.6" 6.3 + 1.3" 10.5 + 1.6"
Human erythrocyte plasma membranes were added with 10 ktM of either of the additions i.e. Spd, Spin, NaF or Forskolin. Data are mean + SD of quadruplicate assay samples. ***The group received 2.5 ~tM of each of all the three Polyamines (Spd, Spm and Put.). Each of the polycations was also used at 10 IxM concentration. Data differ significantly in comparison with control values following to Mann Whitney test of significance *(p < 0.001) ; **(p < 0.01). §Insignificant data in comparison with basal values.
46
Adenylate Cyclase in Human Erythrocytes
Vol. 46, No. i, 1990
The regulatory implications of the observed polyamine induced attenuation of the adenylate cyclase activity in the present study in human erythrocyte plasma membranes remains obscure. However, it might be argued that polyamines might act for self regulatory function. Their systhesis during erythroid maturation of red blood ceils coupled to adenylate cyclase induction of ornithine decarboxylase (ODC), a rate limiting enzyme of polyamine systhesis via the phosphorylation by cAMP dependent kinase would require cAMP for sustained activation. Such a state of affairs necessary to maintain the steady state levels of polyamines has been shown by Caldarera et al (14). They further reported that treatment of primary heart cells with DFMO (alfa difluoromethylornithine), an irreversible inhibitor of ODC activity, resulted in low intracellular polyamine concentrations and high cAMP levels and adenylate cyclase activity (12). It is possible that 10 p.M concentrations of PA may not ordinarily be found in the free state of intact ceils (30), nevertheless, we may propose that the polyamines might play a regulatory role, by altering the activity of second messengers (adenylate cyclase), on the cell surface though the role of polyamines as second messengers has also been suggested by several investigators (31, 32). This idea is further supported by the recent findings of Fan and Koenig (33) who have observed that depletion of intracellular polyamines in heart cells abrogated the stimulation of Ca++ influx, hexose and amino acid transport evoked by isoproterenol. Moreover, polyamines may enhance the phosphorylation of cell proteins following of activation of polyamine dependent protein kinases (34, 35). Further studies are in progress to investigate whether polyamines affect the homologous or heterogolous desensitization of B - adrenergic receptors in chicken erythrocytes and with other cells. It will, in addition, be determined what influence PA have on the generation of cAMP by stimulating Ca++ influx. Acknowledgements This work was supported by research grants from CNRS and a Post Doc Fellowship to one of the authors (NAK) from Association pour Recherche Contre le Cancer (France). 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
References C.E. GUTHROW, J.E. ALLEN and H. RASMUSSEN, J. Biol. Chem. 247, 8145 - 8153 (1972). M.M. HOSEY and M. TAO, Biochemistry. 15, 1561 - 1568 (1976). K.W. SIMKOWSKY and M.TAO, J. Biol. Chem. 255, 6456 - 6461 (1980). P.J.RAVAL and D. ALLEN, Biochem. J. 231, 179 - 183 (1985). J.M. STADEL, B. STRULOVICI, P. NAMBI, T.N. LAVIN, M.M. BRIGGS, M.G. CARON and R.J. LEFKOWITZ, J. Biol. Chem. 258, 3032 - 3038 (1983). D.R. SIBLEY, J.R. PETERS, P. NAMBI, M.G. CARON and R.J. LEFKOWITZ, J. Biol. Chem. 259, 9742 - 9749 (1984). J.M. STADEL, R. REBAR and S.T. CROOKE, Biochem. J. 252, 771 - 776 (1988). K.M. POPOV, T.V. BULARGINA and E.S. SEVERIN, Biokhemiya (Russian).50, 443 446 (1985). L. LEVIA, D. CARRASCO, A. TAYLOR, M. VELIZ, C. GONZALEZ, C.C. ALLENDE and J.E. ALLENDE, Biochem. Int.14, 77-717 (1987). O. TAKEDA, K. YAMASAKI, H. KOHDA, A. YAMASHITA, T. KURIKAWA and S. ISHIBASHI, J. Pharmacobio. - Dyn. 11,377 -380 (1988). C. CLO, C.M. CARDARERA, B. TANTINI, D. BENALAL and U. BACHRACH, Biochem. J. 182, 641 - 649 (1979). C. CLO, C. PIGANATTI, S. MANFRONI, S. MARMIROLI and B. TANTINI, in Biochemical Studies of Natural Polyamines. (eds. C.M. Calderara, C. Clo and C. Guarnieri). CLUEB Bologna, Italy. 117 - 125 (1986). C. CLO, B. TANTINI, C. PIGANATTI, S. MARMIROLI and C.M. CALDARERA, in Persoectives in Polvamine Research. (eds. A. Perin, G. Scalabrino, A. Sessa and M.E. Ferioli).Wichtig Edit-ore, Milano, Italy. 45 - 48 (1988). C.M. CALDARERA, B. TANTINI, S. MARMIROLI, C. PIGNATTI and C. CLO, in .R...gcent Progress in Polvamine Research. (eds. L. Selmeci, M.E. Brosman and N. Seiler) 109 - 119 (1985). R.S KENT, A.D. LEAN and R.J. LEFKOWITZ, Mol. Pharmacol.17, 14 - 23 (1980).
Vol. 46, No. I, 1990
16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.
Adenylate Cyclase in Human Erythrocytes
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