Proc. Natl. Acad. Sci. USAVol. 74, No. 8, pp. 3404-3408, August 1977Cell Biology

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

STEPHEN A. RUDOLPH, PAUL GREENGARD, AND STEPHEN E. MALAWISTADepartments of Pharmacology and Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510

Communicated by Alfred Gilman, May 12, 1977

ABSTRACT The increase in human leukocyte adenosine3':5'-cyclic monophosphate (cyclic AMP) levels seen in responseto various substances was markedly potentiated by colchicineand other agents that affect microtubule assembly. Addition ofdI-isoproterenol (2 AiM) or prostaglandin El (10 ttM), togetherwith the phosphodiesterase inhibitor isobutylmethylxanthine(1 mM), caused a much greater increase in cyclic AMP in col-chicine-pretreated cells tan in control cells. With isoproterenol(2 MM) plus isobutylmethylxanthine (1 mM), cyclic AMP levelsrose about 3fold but, in combination with colchicine, thesedrugs caused a more than 15-fold increase in cyclic AMP. Theeffects of colchicine were both time- and dose-dependent; theycould be seen'within 1 min after addition of colchichme or atconcentrations as low as 10 nM. In addition to its potentiationof hormonally induced increases in cyclic AMP levels, colchi-cine also potentiated the effect of isobutylmethylxanthine aloneon leukocyte cyclic AMP levels. Vinblastine, vincristine, po-dophyllotoxin, and oncodazole all had effects similar to thoseof colchicine but blmicolchicine did not. The data suggest thatcytoplasmic nticrbtubules interact with the leukocyte plasmamembrane to impose constraints on the expression of hor-mone-sensitive adenylate cyclase; the therapeutic effects ofcolchicine may depend in part upon the relaxation of suchconstraints. Moreover, the synergism described here betweencolchicine-like agents and hormones is of potential therapeuticimportanceiln clinical conditions in which either alkaloid orhormone has been useful separately.

I

The cyclic nucleotide levels of human leukocytes (both gran-ulocytes and mononuclear cells) are controlled by a number ofhormones and pharmacological agents (1-5). f3-Adrenergicagonists (isoproterenol, epinephrine), histamine, and prosta-glandin E1 (PGE1) all raise adenosine 3':5'-cyclic monophos-phate (cyclic AMP) levels in these cells; the muscarinic cho-linergic agonists (acetylcholine, carbamylcholine) raise cyclicGMP levels. In granulocytes, a functional significance of cyclicnucleotides has been suggested by experiments showing thatincreases in cyclic AMP inhibit the degranulation of lysosomesthat normally accompanies phagocytosis, whereas increases incyclic GMP enhance degranulation (2, 3, 5, 6). Colchicine andvinblastine, agents that cause the disappearance of cytoplasmicmicrotubules by preventing their assembly (7-10), also inhibitdegranulation (6, 11-13); it is through this inhibition of mi-crotubule assembly, with consequent effects on microtubule-associated functions, that colchicine is thought to exert itstherapeutic anti-inflammatory action in acute gouty arthritisand other disorders (14). We now report that colchicine andother agents that interfere with microtubule assembly increasecyclic AMP levels in human leukocytes and potentiate the ef-fects of hormones on cyclic AMP levels in these cells.

METHODSLeukocytes were obtained from freshly drawn, heparinizedblood from healthy, adult donors by dextran sedimentation andhypotonic lysis of residual erythrocytes as described (11). Theleukocytes were resuspended in 123.5 mM NaCl/5.0 mMKCI/0.3 mM MgCl2/0.5 mM CaCl2/16.0 mM sodium phos-phate/heparin, 1 unit/ml at pH 7.4. Differential counts wereapproximately 70-80% neutrophils and 20-30% lymphocyteswith 1-4% monocytes and eosinophils. Platelets were rare. Allmanipulations were carried out in siliconized glass or plastictubes or flasks.

Cell suspensions (I to 5 X 107 cells per ml) were preincubatedwith colchicine or other drugs, at 370 in a Dubnoff metabolicshaker. Aliquots (200 pl) of the cell suspension were thentransferred to plastic tubes, and incubation was continued foran additional 2 min in the presence of other test substances. Thereaction was terminated by placing the tubes in a boiling waterbath for 5 min. Then, 300 ,l of water was added to each tubeand the samples were frozen. After thawing, the samples werecentrifuged (900 X g for 10 min) to remove particulate mate-rial, and triplicate aliquots were withdrawn (10-100 1d) forcyclic AMP determination. Cyclic AMP was measured by theisotope dilution assay of Brown et al. (15).

Lumicolchicine was prepared by ultraviolet irradiation ofcolchicine solutions as described (16). Conversion was measuredby monitoring absorbance at 267 and 350 nm. Other drugs wereobtained from the following sources: dl-isoproterenol, colchi-cine, 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 fromJ. E. Pike of Upjohn. All chemicals used were reagent grade.

RESULTSThe effect of various concentrations of colchicine on cyclicAMP levels in human leukocytes is shown in Fig. 1. A 30-minpreincubation with maximally effective concentrations ofcolchicine caused a more than 2-fold increase in the cyclic AMPlevel reached after a 2-min incubation with 1 mM iBuMeXan.In the absence of iBuMeXan, the cyclic AMP level was 0.2pmol/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 AmericanSociety for Clinical Investigation, National Meeting, on May 2, 1977,and has appeared in abstract: CGn. Res. 25, 461a, 1977.

3404

The costs of publication of this article were defrayed in part by thepayment of page charges from funds made available to support theresearch which is the subject of the article. This article must thereforebe hereby marked "advertisement" in accordance with 18 U. S. C.§1734 solely to indicate this fact.

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-CellBioogyRuopeProc. NatL.Acad. Sci. USA 74(1977) 3405

1.4

1.2

U 1.0I

0 107 106 lo-, 104Colchicine, M

FIG. 1. Effect of preincubation with various concentrations ofcolchicine on cyclic AMP levels in human leukocytes. Cells werepreincubated for 30 min at 370 in the presence of the indicated con-centrations of colchicine. Aliquots (200 Ml) were withdrawn, trans-ferred to tubes containing 22 M1l of 10mM iBuMeXan, and incubatedfor an additional 2 min. CyclicAMP was then determined as describedin the text. Data shown as mean + SEM (n = 3).

to observe the effects of colchicine. The effects of colchicinewere manifested over a narrow concentration range, with noeffect being observed at O-7M and a virtually maximal effectat 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 dl-isoproterenol (2 MM) in addition to iBuMeXan (1 mM) causeda 4-fold increase in cyclic AMP level over that observed withiBuMeXan alone. Colchicine markedly potentiated the effectsof dl-isoproterenol, causing a 4-fold increase in the response tothe j-adrenergic-agonist. Thus, the combination of preincu-bation with colchicine and treatment with dl-isoproterenol plusiBuMeXan raised cyclic AMP levels more than 15-fold over

10

.8

-E6 ./0E

4-

> 2

0

C - My' . ,0 10-7 10-6 i0-5 104

Coichicine, MFIG. 2. Effect of preincubation with various concentrations of

colchicine on stimulation of cyclic AMP levels by dl-isoproterenol inhuman leukocytes. Cells were preincubated with the indicated con-centrations of colchicine for 30 min at 37°. Aliquots (200 Ml) werewithdrawn, transferred to tubes containing 22 jd of 10mM iBuMeXanplus 2 1sM dl-isoproterenol, and incubated for an additional 2 min (0).Cyclic AMP was then determined as described in the text. The dataof Fig. 1 (no isoproterenol) are presented for comparison (A). Dataare shown as mean + SEM (n = 3).

Table 1.. Effect of colchicine on cyclicAMP levels in humanleukocytes

Cyclic AMP, pmol/106 cells*Colchicine

Additions Control (10 JM)

None 0.39 I 0.02 0.31 I 0.01iBuMeXan, 1 mM 0.74 + 0.03 2.16 t 0.08dI-Isoproterenol, 2 JAM 0.54 :+ 0.04 0.80 + 0.01iBuMeXan + dl-isoproterenol 2.86 ± 0.05 10.8 i 0.7

Cells were preincubated for 30 min at 370 in the absence or presenceof 10MgM colchicine. iBuMeXan or dl-isoproterenol or both were thenadded to the indicated final concentrations, and incubation wascarried out for an additional 2 min; cyclic AMP was then determinedas described in the text.* Mean SEM.

those observed in the presence of iBuMeXan alone. This syn-ergism of- colchicine and dl-isoproterenol developed over anarrow 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 re-

quired for detecting maximal effects of colchicine and dl-iso-proterenol, 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 colchicineconcentration shown (I0-7 M), there was no significant effectbefore 60 min of preincubation, but there was-a 3fold increaseat 120 min. With the highest concentration shown (10-6 M),there was a half-maximal increase in cyclic AMP level within20 min and a maximal increase at 30 min. With 5 X 10-5 Mcolchicine there was a half-maximal increase within 1 min anda maximal effect within 2 min (data not shown). Thus, thecolchicine dose-response curves shown in Figs. 1 and 2 (30-minpreincubation with colchicine) are time-dependent and wouldbe shifted to the left with increasing time of incubation. Thelowest concentration of colchicine tested was 10-8 M (notshown); after a 3-hr preincubation, the potentiation of the effectof isoproterenol plus iBuMeXan was about half that observedwith maximally effective concentrations of colchicine. Thetime-dependence of the colchicine effect could be due to lim-itation of the rate of entry of the drug into the cells or to limi-tation of- the rate of its binding to tubulin.In order to investigate further the relationship between the

'0 10 20 30 40 50 60 70 80 90 100 110 120Preincubation with colchicine, min

FIG. 3. Time- and dose-dependence of the effect of colchicineon isoproterenol-stimulated cyclic AMP level in human leukocytes.Cells were preincubated with the indicated concentrations of col-chicine. At the times shown, 200-gl aliquots were withdrawn, trans-ferred to tubes containing 22 Mi of 10 mM iBuMeXan plus 20 ,Mdl-isoproterenol, and incubated for an additional 2 min. Cyclic AMPwas then determined as described in the teit.

. Cell Biology: Rudolph et al.

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3406 Cell Biology: Rudolph et al.

d/-lsoprote , M ,uMX45, min

25-

FIG. 4. Effcts o dl-CoichicineE20 ~50MM X

A lur45 mi

then added to a final concentration of 1 with the indiControlControl

iO1-1 i0-1 10-7 10-6 i0-5 -0-9 lo-, lo-7 10 6 lo-,

d/-isoproterenol, M PGE,, M

FIG. 4. Effects of dl-isoproterenol, PGE1, and colchicine on cyclicAMP levels in human leukocytes. Cells were preincubated for 45 mnat 370 in the absence or presence of 50,MM colchicine. iBuMeXan was

then added to a final concentration of 1 mM with the indicated finalconcentrations 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 + colchi-

cine; PGEr; n,PGE c+ colchicine.

effects of colchicine and hormonal stimulation on leukocyte

cyclic AMP levels, dose-response curves for dl-isoproterenoland PGEI were determined in the presence and absence of

colchicine. The results are shown in Fig. 4. In the absence of

colchicine, both dl-isoproterenol andPGE) caused significantincreases in cyclic AMP levels (6-fold and 12-fold, respectively),and these effects were potentiated by preincubation with col-

chicine. The concentrations of dl-isoproterenol andPGEe atwhich stimulation was first observed(10f8 and10sM, re-

spectively) were not affected by colchicine, nor were the con-

centrations for half-maximal and maximal stimulation by dl-isoproterenol. The increase in cyclic AMP levels elicited byPGEi did not appear to be maximal even at concentrations as

high as 10i oM PGE1 (not shown).In some experiments, leukocyte preparations were separated

into granulocyte and mononuclear cell-rich fractions by cen-

trifugation 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 bydl-isoproterenol. These results

are shown in Table 2. Vinblastine, vincrist(ne, oncodazole, and

podophyllotoxin all were effective (as was colchicine) in po-tentiating 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 cytoplasmicmicrotubules. Lumicolchicine, at the same concentration (10

,gM) and incubation time (30 min), was without effect. Cyto-chalasin B, an agent known to interfere with microfilament

structure, had little or no effect on either resting or hormonallystimulated cyclic AMP levels.

It has been reported that colchicine may stimulate prosta-

glandin release from cultured cells (19). It is unlikely that this

effect might account for the increased cyclic AMP levels ob-

served in colchicine-treated leukocytes, because neither in-

domethacin (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

Table 2. Effect of various drugs on leukocyte cyclic AMP levels

Cyclic AMP, pmol/106 cells*Additions Control Isoproterenol

None 0.74 ± 0.03 2.86 ± 0.05Colchicine, 10MM 2.16 + 0.08 10.8 + 0.7Vinblastine, 10,vM 2.35 i 0.10 13.1 ± 0.1Vincristine, 10 MM 2.41 ± 0.18 14.4 i 0.4Oncodazole, 10 MM 1.55 i 0.07 13.9 i 0.1Podophyllotoxin, 10 MM 1.76 + 0.13 12.9 i 0.5Cytochalasin B, 10,MM 0.77 i 0.07 3.16 ± 0.36Lumicolchicine, 10,MM 0.73 i 0.10 2.40 ± 0.02Indomethacin, 100 MM 0.68 ± 0.03 2.50 i 0.10Indomethacin + colchicine 1.94 + 0.12 9.57 + 0.22Aspirin, 1 mM 0.84 ± 0.05 3.08 ± 0.03Aspirin + colchicine 1.95 i 0.10 10.9 + 0.2Hydrocortisone, 1 mM 0.84 ± 0.09 2.59 + 0.14Sodium gold thiomalate, 1.3 mM 0.79 ± 0.03 3.33 ± 0.41Chloroquine, 10 MM 0.77 ± 0.03 2.55 i 0.17

Cells were preincubated for 30 min at 370 in the presence of theindicated drugs. Incubation was then carried out for an additional 2min in the presence of iBuMeXan alone (control) or plus dl- isopro-terenol. The final concentrations of iBuMeXan and of dl- isoproter-enol were 1 mM and 2 MM, respectively. Cyclic AMP was determinedas described in the text. Dimethyl sulfoxide was used to dissolve on-codazole, podophyllotoxin, and cytochalasin B. Ethanol was used todissolve indomethacin and aspirin. These solvents were present ata final concentration of 1% in incubations with the above drugs; nei-ther dimethyl sulfoxide (1%) nor ethanol (1%) affected cyclic AMPlevels under any of the experimental conditions tested.* Mean i SEM (n = 3).

when present during the 30-min preincubation period. Hy-drocortisone (1 mM), gold sodium thiomalate (1.3 mM), andchloroquine (10 AuM) also had no effect.

DISCUSSIONThe data presented show that colchicine and other agents thatinterfere with microtubule assembly cause an increase inhuman leukocyte cyclic AMP levels; these agents also potentiate0B-adrenergic and PGE1 stimulation of cyclic AMP levels. Theseeffects of colchicine could be manifested through an increasein the rate of production of cyclic AMP or a decrease in its rateof hydrolysis. The latter possibility, however, seems unlikely,because a phosphodiesterase inhibitor (iBuMeXan) is requiredfor the effects of colchicine to be detected; preincubation of thecells with colchicine alone is relatively ineffective in stimulatingcyclic AMP levels, in either the absence or the presence ofhormones. Therefore, it would appear that colchicine acts toincrease adenylate cyclase activity. Such an increase might beaccomplished in any of several ways: (i) the availability ofsubstrate (ATP) might be increased; (ii) the intrinsic turnovernumber of the enzyme might be increased; (iii) the number ofactive adenylate cyclase molecules might be increased; (iv) thehormone receptor-adenylate cyclase interaction might be mademore efficient; or (v) the guanyl nucleotide binding site orguanyl nucleotide metabolism might be affected.The data shown in Table 2 indicate that other known in-

hibitors of microtubule assembly (vinblastine, vincristine, on-codazole, and podophyllotoxin) have effects similar to those ofcolchicine, whereas lumicolchicine, which does not inhibit as-sembly, has no such effect. These chemically distinct agentshave in common the ability to bind to tubulin; that they do notall bind at the same site is further evidence for their specificityof action (17, 20, 21). It thus seems probable that the effects of

Proc. Natl. Acad. Sci. USA 74 (1977)

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Cell Biology: Rudolph et al.

colchicine are a result of its inhibition of tcrotubuleMoreover, the effects of preincubation with colchicine do notpersist after the cells are broken, nor does colchicine affect di-rectly either basal or hormone-stimulated adenylate cyclaseactivity in leukocyte membrane preparations (data not shown),even though these preparations respond readily to the hormonesthemselves, suggesting that the colchicine effect on cyclic AMPlevels is a result of its interaction with cytoplasmic, rather thanmembrane-associated, tubulin.

It has been suggested that colchicine-sensitive cytoplasmicstructures (presumably microtubules) may interact with the cellmembrane in such a way as to place constraints on the mobilityof certain receptors (22-25). Previous work has involved pri-marily lectin-binding receptors. It is interesting to speculate onhow such an interaction might be responsible for effects onreceptor-mediated changes in cyclic AMP reported here. Asshown in Fig. 4, colchicine increased both basal and hor-mone-stimulated levels of cyclic AMP, and we have interpretedthese effects as being due to an increase in adenylate cyclaseactivity. Current evidence suggests that hormone receptors andadenylate cyclase are separate entities (26) and that the receptorinteracts with the adenylate cyclase to cause activation (27); theapparent affinity of this interaction is greatly enhanced whenthe receptor is occupied by the appropriate hormone. Thus, thetime of interaction between receptor and cyclase will be longerwhen the receptor is occupied. In order to facilitate discussionof this point, we can express the total adenylate cyclase activityas follows:

(nac,- Tac) + (Nr-ac tr-ac Tr-ac)(Nrh-ac' trh ac' Trh-a)

in which n, = number of adenylate cyclase molecules notcomplexed with receptors; TaC = intrinsic turnover number ofadenylate cyclase in the absence of receptor; N, = frequencyof unoccupied receptor-adenylate cyclase interactions; t,.ac= average duration of unoccupied receptor-adenylate cyclaseinteraction; T7 as = turnover number of unoccupied recep-tor-adenylate cyclase complex; Nrha = frequency of occupiedreceptor-adenylate cyclase interactions; trh a = average du-ration of occupied receptor-adenylate cyclase interaction; andTrh-ac = turnover number of occupied receptor-adenylatecyclase complex.

If the effect of colchicine is to increase the lateral mobilityof cell surface receptors by relaxing the constraints imposed byinteractions between cytoplasmic microtubules and the plasmamembrane, then we would expect the frequency of encounterbetween receptors and adenylate cyclase molecules to increasein colchicine-treated cells. This would lead to an increase in theterms Nr and Nrh , giving an increase in both basal andhormone-stimulated adenylate cyclase activity. With this in-terpretation, the increase in leukocyte cyclic AMP levels causedby colchicine would not be due to a specific effect on either thereceptor or the adenylate cyclase but rather to an increase inplasma membrane mobility, resulting in more frequent inter-action among plasma membrane components. Although thedata presented here do not support this conclusion unambigu-ously, they are consistent with it and with previous interpre-tations of the effects of colchicine on events mediated by cellsurface receptors.

It will be of interest to look for functional concomitants ofthe synergistic relationship between colchicine and hormonesreported here. There is already some evidence for their exis-tence. For example, the effect of colchicine in melanocytes isamplified by agents that act rapidly and reversibly on granule

Proc. Nati. Acad. Sci. USA 74 (1977) 3407

Version, including those acting through cyclic AMP, suchas melanocyte-stimulating hormone and caffeine (28). Otherantimitotic 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, isattained in plasma with doses of the drug used therapeutically(31). If functional synergism between colchicine-like drugs andthose hormones whose effects are mediated through cyclic AMPis a more general phenomenon, then appropriate combinationsof agents may provide increased therapeutic power in situationsin which either class of drugs has proven useful but often notideal when used alone-for example, colchicine, used as ananti-inflammatory or antimitotic agent, and fl-adrenergicagonists, used in allergic and asthmatic disorders.

This work was supported by U.S. Public Health Service GrantsMH-17387 and NS-8440 to P.G. and U.S. Public Health Service GrantsAM-10493, AM-19742, AM-05639, and AM-07107 and grants fromthe Arthritis Foundation and the Kroc Foundation to S.E.M.

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21. Wilson, L. (1975) "Microtubules as drug receptors: pharmaco-logical properties of microtubule protein," Ann. N.Y. Acad. Sci.253,213-231.

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Proc. Natl. Acad. Sci. USA 74 (1977)

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