Effects of colchicine on cyclic AMP levels in human leukocytes* (microtubules/adenylate cyclase/catecholamines/prostaglandins)

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 74, No. 8, pp , August 1977 Cell Biology Effects of colchicine on cyclic AMP levels in human leukocytes* (microtubules/adenylate cyclase/catecholamines/prostaglandins) STEPHEN A. RUDOLPH, PAUL GREENGARD, AND STEPHEN E. MALAWISTA Departments of Pharmacology and Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut Communicated by Alfred Gilman, May 12, 1977 ABSTRACT The increase in human leukocyte adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels seen in response to various substances was markedly potentiated by colchicine and other agents that affect microtubule assembly. Addition of di-isoproterenol (2 AiM) or prostaglandin El (10 ttm), together with the phosphodiesterase inhibitor isobutylmethylxanthine (1 mm), caused a much greater increase in cyclic AMP in colchicine-pretreated cells tan in control cells. With isoproterenol (2 MM) plus isobutylmethylxanthine (1 mm), cyclic AMP levels rose about 3fold but, in combination with colchicine, these drugs caused a more than 15-fold increase in cyclic AMP. The effects of colchicine were both time- and dose-dependent; they could be seen'within 1 min after addition of colchichme or at concentrations as low as 10 nm. In addition to its potentiation of hormonally induced increases in cyclic AMP levels, colchicine also potentiated the effect of isobutylmethylxanthine alone on leukocyte cyclic AMP levels. Vinblastine, vincristine, podophyllotoxin, and oncodazole all had effects similar to those of colchicine but blmicolchicine did not. The data suggest that cytoplasmic nticrbtubules interact with the leukocyte plasma membrane to impose constraints on the expression of hormone-sensitive adenylate cyclase; the therapeutic effects of colchicine may depend in part upon the relaxation of such constraints. Moreover, the synergism described here between colchicine-like agents and hormones is of potential therapeutic importanceiln clinical conditions in which either alkaloid or hormone has been useful separately. I The cyclic nucleotide levels of human leukocytes (both granulocytes and mononuclear cells) are controlled by a number of hormones and pharmacological agents (1-5). f3-adrenergic agonists (isoproterenol, epinephrine), histamine, and prostaglandin E1 (PGE1) all raise adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in these cells; the muscarinic cholinergic agonists (acetylcholine, carbamylcholine) raise cyclic GMP levels. In granulocytes, a functional significance of cyclic nucleotides has been suggested by experiments showing that increases in cyclic AMP inhibit the degranulation of lysosomes that normally accompanies phagocytosis, whereas increases in cyclic GMP enhance degranulation (2, 3, 5, 6). Colchicine and vinblastine, agents that cause the disappearance of cytoplasmic microtubules by preventing their assembly (7-10), also inhibit degranulation (6, 11-13); it is through this inhibition of microtubule assembly, with consequent effects on microtubuleassociated functions, that colchicine is thought to exert its therapeutic anti-inflammatory action in acute gouty arthritis and other disorders (14). We now report that colchicine and other agents that interfere with microtubule assembly increase cyclic AMP levels in human leukocytes and potentiate the effects of hormones on cyclic AMP levels in these cells. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact. METHODS Leukocytes were obtained from freshly drawn, heparinized blood from healthy, adult donors by dextran sedimentation and hypotonic lysis of residual erythrocytes as described (11). The leukocytes were resuspended in mm NaCl/5.0 mm KCI/0.3 mm MgCl2/0.5 mm CaCl2/16.0 mm sodium phosphate/heparin, 1 unit/ml at ph 7.4. Differential counts were approximately 70-80% neutrophils and 20-30% lymphocytes with 1-4% monocytes and eosinophils. Platelets were rare. All manipulations were carried out in siliconized glass or plastic tubes or flasks. Cell suspensions (I to 5 X 107 cells per ml) were preincubated with colchicine or other drugs, at 370 in a Dubnoff metabolic shaker. Aliquots (200 pl) of the cell suspension were then transferred to plastic tubes, and incubation was continued for an additional 2 min in the presence of other test substances. The reaction was terminated by placing the tubes in a boiling water bath for 5 min. Then, 300,l of water was added to each tube and the samples were frozen. After thawing, the samples were centrifuged (900 X g for 10 min) to remove particulate material, and triplicate aliquots were withdrawn ( d) for cyclic AMP determination. Cyclic AMP was measured by the isotope dilution assay of Brown et al. (15). Lumicolchicine was prepared by ultraviolet irradiation of colchicine solutions as described (16). Conversion was measured by monitoring absorbance at 267 and 350 nm. Other drugs were obtained from the following sources: dl-isoproterenol, colchicine, chloroquine, and isobutylmethylxanthine (ibumexan) from Sigma; vinblastine (Velban) and vincristine (Oncovin) from Eli Lilly; podophyllotoxin from Aldrich; oncodazole [R-17934, a new agent that inhibits microtubule assembly (17)] from Janssen; cytochalasin B from ICN; sodium gold thiomalate (Myochrysine) from Merck, Sharp and Dohme; hydrocortisone (Solu-Cortef) from Upjohn. Prostaglandin E1 was a gift from J. E. Pike of Upjohn. All chemicals used were reagent grade. RESULTS The effect of various concentrations of colchicine on cyclic AMP levels in human leukocytes is shown in Fig. 1. A 30-min preincubation with maximally effective concentrations of colchicine caused a more than 2-fold increase in the cyclic AMP level reached after a 2-min incubation with 1 mm ibumexan. In the absence of ibumexan, the cyclic AMP level was 0.2 pmol/106 cells in both control and colchicine-treated cells. Addition of the phosphodiesterase inhibitor was thus necessary Abbreviations: PGE1, prostaglandin E1; cyclic AMP, adenosine 3':5'- cyclic monophosphate; ibumexan, isobutylmethylxanthine. * A preliminary report of this work was presented at the American Society for Clinical Investigation, National Meeting, on May 2, 1977, and has appeared in abstract: CGn. Res. 25, 461a,

2 Cell- BioogyRuopeProc. NatL.Acad. Sci. USA 74(1977) Cell Biology: Rudolph et al U 1.0I lo-, 104 Colchicine, M FIG. 1. Effect of preincubation with various concentrations of colchicine on cyclic AMP levels in human leukocytes. Cells were preincubated for 30 min at 370 in the presence of the indicated concentrations of colchicine. Aliquots (200 Ml) were withdrawn, transferred to tubes containing 22 M1l of 10 mm ibumexan, and incubated for an additional 2 min. Cyclic AMP was then determined as described in the text. Data shown as mean + SEM (n = 3). to observe the effects of colchicine. The effects of colchicine were manifested over a narrow concentration range, with no effect being observed at O-7 M and a virtually maximal effect at 10-6 M. The effect of preincubation with colchicine was also tested on the isoproterenol-induced increase in cyclic AMP level (Fig. 2). In the absence of colchicine, incubation for 2 min with dlisoproterenol (2 MM) in addition to ibumexan (1 mm) caused a 4-fold increase in cyclic AMP level over that observed with ibumexan alone. Colchicine markedly potentiated the effects of dl-isoproterenol, causing a 4-fold increase in the response to the j-adrenergic-agonist. Thus, the combination of preincubation with colchicine and treatment with dl-isoproterenol plus ibumexan raised cyclic AMP levels more than 15-fold over 10.8 Table 1.. Effect of colchicine on cyclic AMP levels in human leukocytes Cyclic AMP, pmol/106 cells* Colchicine Additions Control (10 JM) None 0.39 I I 0.01 ibumexan, 1 mm t 0.08 di-isoproterenol, 2 JAM 0.54 : ibumexan + dl-isoproterenol 2.86 ± i 0.7 Cells were preincubated for 30 min at 370 in the absence or presence of 10MgM colchicine. ibumexan or dl-isoproterenol or both were then added to the indicated final concentrations, and incubation was carried out for an additional 2 min; cyclic AMP was then determined as described in the text. * Mean SEM. those observed in the presence of ibumexan alone. This synergism of- colchicine and dl-isoproterenol developed over a narrow colchicine concentration range (2 X 10-7 to 1 X 10- M), similar to that effective in the absence of isoproterenol. As shown in Table 1, a phosphodiesterase inhibitor was required for detecting maximal effects of colchicine and dl-isoproterenol, either alone or in combination. The time dependence of the effect of colchicine on leukocyte cyclic AMP level is shown in Fig. 3. At the lowest colchicine concentration shown (I0-7 M), there was no significant effect before 60 min of preincubation, but there was-a 3fold increase at 120 min. With the highest concentration shown (10-6 M), there was a half-maximal increase in cyclic AMP level within 20 min and a maximal increase at 30 min. With 5 X 10-5 M colchicine there was a half-maximal increase within 1 min and a maximal effect within 2 min (data not shown). Thus, the colchicine dose-response curves shown in Figs. 1 and 2 (30-min preincubation with colchicine) are time-dependent and would be shifted to the left with increasing time of incubation. The lowest concentration of colchicine tested was 10-8 M (not shown); after a 3-hr preincubation, the potentiation of the effect of isoproterenol plus ibumexan was about half that observed with maximally effective concentrations of colchicine. The time-dependence of the colchicine effect could be due to limitation of the rate of entry of the drug into the cells or to limitation of- the rate of its binding to tubulin. In order to investigate further the relationship between the -E6./ 0 E - 4 > 2 0 FIG. 2. C - My'., i Coichicine, M Effect of preincubation with various concentrations of colchicine on stimulation of cyclic AMP levels by dl-isoproterenol in human leukocytes. Cells were preincubated with the indicated concentrations of colchicine for 30 min at 37. Aliquots (200 Ml) were withdrawn, transferred to tubes containing 22 jd of 10 mm ibumexan plus 2 1sM dl-isoproterenol, and incubated for an additional 2 min (0). Cyclic AMP was then determined as described in the text. The data of Fig. 1 (no isoproterenol) are presented for comparison (A). Data are shown as mean + SEM (n = 3). ' Preincubation with colchicine, min FIG. 3. Time- and dose-dependence of the effect of colchicine on isoproterenol-stimulated cyclic AMP level in human leukocytes. Cells were preincubated with the indicated concentrations of colchicine. At the times shown, 200-gl aliquots were withdrawn, transferred to tubes containing 22 Mi of 10 mm ibumexan plus 20,M dl-isoproterenol, and incubated for an additional 2 min. Cyclic AMP was then determined as described in the teit.

3 3406 Cell Biology: Rudolph et al. Proc. Natl. Acad. Sci. USA 74 (1977) Table 2. Effect of various drugs on leukocyte cyclic AMP levels 25- d/-lsoprote, M,uMX45, min FIG. 4. Effcts o dl-coichicine E20 ~50 MM X A lur 45 mi then added to a final concentration of 1 Control with the indicontrol io1-1 i i lo-, lo lo-, d/-isoproterenol, M PGE,, M FIG. 4. Effects of dl-isoproterenol, PGE1, and colchicine on cyclic AMP levels in human leukocytes. Cells were preincubated for 45 mn at 370 in the absence or presence of 50,MM colchicine. ibumexan was then added to a final concentration of 1 mm with the indicated final concentrations of either di-isoproterenol (Left) or PGe1 (Right). After an additional 2-mmvincubation, cyclic AMP was determined as described in the text. 0, Isoproterenol; o, isoproterenol + colchicine; PGEr; n,pge + colchicine. effects of colchicine and hormonal stimulation on leukocyte cyclic AMP levels, dose-response curves for dl-isoproterenol were determined in the presence and absence of and PGEI colchicine. The results are shown in Fig. 4. In the absence of colchicine, both dl-isoproterenol PGE) and caused significant increases in cyclic AMP levels (6-fold and 12-fold, respectively), and these effects were potentiated by preincubation with colchicine. The concentrations of dl-isoproterenol and PGEe at which stimulation was first observed(10f8 and 10sM, respectively) were not affected by colchicine, nor were the concentrations for half-maximal and maximal stimulation by dlisoproterenol. The increase in cyclic AMP levels elicited by PGEi did not appear to be maximal even at concentrations as high as 10i M PGE1 o (not shown). In some experiments, leukocyte preparations were separated into granulocyte and mononuclear cell-rich fractions by centrifugation through Ficoll-Hypaque (18). Similar effects of ibumexan, colchicine, isoproterenol, and PGEj, either alone or in combination, were seen in both fractions. Various other pharmacological agents were also tested for their effects on leukocyte cyclic AMP levels, both with and without hormonal stimulation by dl-isoproterenol. These results are shown in Table 2. Vinblastine, vincrist(ne, oncodazole, and podophyllotoxin all were effective (as was colchicine) in potentiating the effects of ibumexan (1 mm) and of ibumexan plus dl-isoproterenol (2 stm) on cyclic AMP levels. All of these agents are known to interfere with the assembly of cytoplasmic microtubules. Lumicolchicine, at the same concentration (10,gM) and incubation time (30 min), was without effect. Cytochalasin B, an agent known to interfere with microfilament structure, had little or no effect on either resting or hormonally stimulated cyclic AMP levels. It has been reported that colchicine may stimulate prostaglandin release from cultured cells (19). It is unlikely that this effect might account for the increased cyclic AMP levels observed in colchicine-treated leukocytes, because neither indomethacin (0. 1 mm) nor aspirin (1 mm), both of which are potent inhibitors of prostaglandin synthesis, had any effect on the colchicine-mediated stimulation of cyclic AMP levels, even Cyclic AMP, pmol/106 cells* Additions Control Isoproterenol None 0.74 ± ± 0.05 Colchicine, 10MM Vinblastine, 10,vM 2.35 i ± 0.1 Vincristine, 10 MM 2.41 ± i 0.4 Oncodazole, 10 MM 1.55 i i 0.1 Podophyllotoxin, 10 MM i 0.5 Cytochalasin B, 10,MM 0.77 i ± 0.36 Lumicolchicine, 10,MM 0.73 i ± 0.02 Indomethacin, 100 MM 0.68 ± i 0.10 Indomethacin + colchicine Aspirin, 1 mm 0.84 ± ± 0.03 Aspirin + colchicine 1.95 i Hydrocortisone, 1 mm 0.84 ± Sodium gold thiomalate, 1.3 mm 0.79 ± ± 0.41 Chloroquine, 10 MM 0.77 ± i 0.17 Cells were preincubated for 30 min at 370 in the presence of the indicated drugs. Incubation was then carried out for an additional 2 min in the presence of ibumexan alone (control) or plus dl- isoproterenol. The final concentrations of ibumexan and of dl- isoproterenol were 1 mm and 2 MM, respectively. Cyclic AMP was determined as described in the text. Dimethyl sulfoxide was used to dissolve oncodazole, podophyllotoxin, and cytochalasin B. Ethanol was used to dissolve indomethacin and aspirin. These solvents were present at a final concentration of 1% in incubations with the above drugs; neither dimethyl sulfoxide (1%) nor ethanol (1%) affected cyclic AMP levels under any of the experimental conditions tested. * Mean i SEM (n = 3). when present during the 30-min preincubation period. Hydrocortisone (1 mm), gold sodium thiomalate (1.3 mm), and chloroquine (10 AuM) also had no effect. DISCUSSION The data presented show that colchicine and other agents that interfere with microtubule assembly cause an increase in human leukocyte cyclic AMP levels; these agents also potentiate 0B-adrenergic and PGE1 stimulation of cyclic AMP levels. These effects of colchicine could be manifested through an increase in the rate of production of cyclic AMP or a decrease in its rate of hydrolysis. The latter possibility, however, seems unlikely, because a phosphodiesterase inhibitor (ibumexan) is required for the effects of colchicine to be detected; preincubation of the cells with colchicine alone is relatively ineffective in stimulating cyclic AMP levels, in either the absence or the presence of hormones. Therefore, it would appear that colchicine acts to increase adenylate cyclase activity. Such an increase might be accomplished in any of several ways: (i) the availability of substrate (ATP) might be increased; (ii) the intrinsic turnover number of the enzyme might be increased; (iii) the number of active adenylate cyclase molecules might be increased; (iv) the hormone receptor-adenylate cyclase interaction might be made more efficient; or (v) the guanyl nucleotide binding site or guanyl nucleotide metabolism might be affected. The data shown in Table 2 indicate that other known inhibitors of microtubule assembly (vinblastine, vincristine, oncodazole, and podophyllotoxin) have effects similar to those of colchicine, whereas lumicolchicine, which does not inhibit assembly, has no such effect. These chemically distinct agents have in common the ability to bind to tubulin; that they do not all bind at the same site is further evidence for their specificity of action (17, 20, 21). It thus seems probable that the effects of

4 Cell Biology: Rudolph et al. colchicine are a result of its inhibition of tcrotubule Moreover, the effects of preincubation with colchicine do not persist after the cells are broken, nor does colchicine affect directly either basal or hormone-stimulated adenylate cyclase activity in leukocyte membrane preparations (data not shown), even though these preparations respond readily to the hormones themselves, suggesting that the colchicine effect on cyclic AMP levels is a result of its interaction with cytoplasmic, rather than membrane-associated, tubulin. It has been suggested that colchicine-sensitive cytoplasmic structures (presumably microtubules) may interact with the cell membrane in such a way as to place constraints on the mobility of certain receptors (22-25). Previous work has involved primarily lectin-binding receptors. It is interesting to speculate on how such an interaction might be responsible for effects on receptor-mediated changes in cyclic AMP reported here. As shown in Fig. 4, colchicine increased both basal and hormone-stimulated levels of cyclic AMP, and we have interpreted these effects as being due to an increase in adenylate cyclase activity. Current evidence suggests that hormone receptors and adenylate cyclase are separate entities (26) and that the receptor interacts with the adenylate cyclase to cause activation (27); the apparent affinity of this interaction is greatly enhanced when the receptor is occupied by the appropriate hormone. Thus, the time of interaction between receptor and cyclase will be longer when the receptor is occupied. In order to facilitate discussion of this point, we can express the total adenylate cyclase activity as follows: (nac,- Tac) + (Nr-ac tr-ac Tr-ac) (Nrh-ac' trh ac' Trh-a) in which n, = number of adenylate cyclase molecules not complexed with receptors; TaC = intrinsic turnover number of adenylate cyclase in the absence of receptor; N, = frequency of unoccupied receptor-adenylate cyclase interactions; t,.ac = average duration of unoccupied receptor-adenylate cyclase interaction; T7 as = turnover number of unoccupied receptor-adenylate cyclase complex; Nrha = frequency of occupied receptor-adenylate cyclase interactions; trh a = average duration of occupied receptor-adenylate cyclase interaction; and Trh-ac = turnover number of occupied receptor-adenylate cyclase complex. If the effect of colchicine is to increase the lateral mobility of cell surface receptors by relaxing the constraints imposed by interactions between cytoplasmic microtubules and the plasma membrane, then we would expect the frequency of encounter between receptors and adenylate cyclase molecules to increase in colchicine-treated cells. This would lead to an increase in the terms Nr and Nrh, giving an increase in both basal and hormone-stimulated adenylate cyclase activity. With this interpretation, the increase in leukocyte cyclic AMP levels caused by colchicine would not be due to a specific effect on either the receptor or the adenylate cyclase but rather to an increase in plasma membrane mobility, resulting in more frequent interaction among plasma membrane components. Although the data presented here do not support this conclusion unambiguously, they are consistent with it and with previous interpretations of the effects of colchicine on events mediated by cell surface receptors. It will be of interest to look for functional concomitants of the synergistic relationship between colchicine and hormones reported here. There is already some evidence for their existence. For example, the effect of colchicine in melanocytes is amplified by agents that act rapidly and reversibly on granule Proc. Nati. Acad. Sci. USA 74 (1977) 3407 Version, including those acting through cyclic AMP, such as melanocyte-stimulating hormone and caffeine (28). Other antimitotic agents have effects similar to those of colchicine (29), whereas lumicolchicine does not (30). The lowest concentration of colchicine tested in the present study (10-8 M), which was effective after a 3-hr incubation, is attained in plasma with doses of the drug used therapeutically (31). 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