phorbol myristate acetate stimulation of lymphocyte ......cyclic gmp phosphodiesterase. 35...

[CANCER RESEARCH 43. 150-1 58. January 1983]0008-5472/83/0043-OOOOS02.00

Phorbol Myristate Acetate Stimulation of Lymphocyte Guanylate Cyclaseand Cyclic GuarÃ-osme3':5'-Monophosphate Phosphodiesterase andReduction of Adenylate Cyclase1

Ronald G. Coffey2 and John W. Madden

Department of Pharmacology. University of South Florida College of Medicine. Tampa. Florida 33612

ABSTRACT

Human peripheral blood lymphocytes incubated with thetumor promoter and T-cell mitogen phorbol-12-myristate-13-acetate (PMA) had elevated cyclic guanosine 3':5'-monophos-

phate (cyclic GMP) levels and activities of guanylate cyclaseand cyclic GMP phosphodiesterase and reduced activity ofadenylate cyclase. No changes occurred in cyclic adenosine3':5'-monophosphate levels or cyclic adenosine 3':5'-mono-

phosphate phosphodiesterase. Cyclic GMP levels increased ina bimodal fashion corresponding to the brisk rise at 1 min inboth membrane and soluble guanylate cyclases and to a second increase at 20 to 60 min in soluble guanylate cyclase.Decreases in adenylate cyclase were similar for basal andisoproterenol- or NaF-stimulated activity. The magnitude of

these changes increased with increasing concentrations ofPMA from 0.01 to 10 /ig/ml, while the effects of PMA onlymphocyte proliferation progressively increase from 0.001 to1 /ig/ml. The effects of a series of phorbol diesters on guanylate cyclase were consistent with their relative activities forlymphocyte mitogenesis and for tumor promotion. Because themononuclear cell populations are heterogeneous, cause andeffect relationships between enzyme changes and biologicalresponses cannot now be ascertained. None of the PMA-in-

duced changes were significantly affected by inhibition ofmacromolecular synthesis or by omitting Ca2+ from the lym

phocyte preincubation medium. PMA-induced increases in

both membrane and soluble guanylate cyclase were markedlyinhibited by the intracellular Ca2+ antagonist methoxyverapamilbut not by [ethylene glycol bis(/?-aminoethyl ether)-N,N,N',N'-

tetraacetic acid. PMA stimulation of the membrane enzymewas prevented by the arachidonic acid lipoxygenase inhibitors15-hydroxyeicosatetraenoic acid and nordihydroguaiaretic

acid. Increases in soluble guanylate cyclase and cyclic GMPphosphodiesterase and reduction of adenylate cyclase by PMAwere less susceptible to the lipoxygenase inhibitors. Theseresults suggest that PMA stimulates cyclic GMP synthesis bya Ca2+-dependent process which, in the membrane, involves

one or more lipoxygenase metabolites and that different mechanisms may account for the alterations in the other cyclicnucleotide-metabolizing enzyme activities.

INTRODUCTION

The human blood lymphocyte represents an important modelfor studying the early events following growth stimulation by

1This study was supported in part by Grants BC 216B from the American

Cancer Society. CA 08748 from the NIH, and by Newport Pharmaceuticals,Newport, Calif. A preliminary report of some of this work was presented at the65th Annual Meeting of the Federation of American Societies for ExperimentalBiology, St. Louis, Mo.. 1981 (10).

2 To whom requests for reprints should be addressed.

Received May 6, 1982; accepted October 8. 1982.

lectin mitogens such as PHA3 and concanavalin A. The plant

terpene diester PMA acts as a tumor promoter in the 2-stage

epidermal carcinogenesis model (2) and is also a potent mitogen for human lymphocytes (18, 56) as well as several animalcell culture systems (14, 26, 63). While PMA acts as a comi-togen in mouse (1), guinea pig (51), and bovine (61) lymphocytes, it is a complete mitogen in human (1, 18) and otherprimate (1) peripheral blood T-lymphocytes. PMA has been

further characterized (18, 56) as selective for a subpopulationof thymus-dependent lymphocytes that is distinct from the

subpopulations responsive to PHA or concanavalin A andwhich may include T-suppressor cells (22, 40). On the other

hand, PMA has been reported to stimulate DNA synthesis inhuman B-cells (1) and, with the cooperation of T-cells, toinduce immunoglobulin secretion in human B-cell lines (46).

While the lectin mitogen stimulation is thought to require accessory cells (51), the growth-stimulating activity of PMA inhuman T-lymphocytes appears to be free of accessory cell

dependence (52) and perhaps acts through direct induction ofinterleukin 2 release (20, 51). PMA, therefore, offers a usefulprobe of the mechanisms of lymphocyte activation.

Lectin mitogens bind to cell surface receptors and need notenter the cell to trigger cell activation (24). PMA also binds withhigh affinity to human lymphocyte receptors and is not metabolized significantly for at least 1 hr (16, 52). Thus, the primarysite of action of PMA in lymphocytes (16-18) and other cells(3, 53) is believed to be at the cell surface, activating a seriesof biochemical changes including turnover of phospholipids (3,36), arachidonic acid production (6), and transport processes(25, 53).

Alterations in cyclic nucleotide metabolism have been foundin association with many of these effects of PMA. Rapid increases in the levels of cyclic GMP have been reported following PMA addition in a number of cell culture systems (5, 18,25, 26, 30, 44, 49, 57, 63). Some workers have found decreases in cyclic AMP (4, 25, 45, 63, 65), while others (5, 38,41, 48) reported an uncoupling of adenylate cyclase fromhormone receptor stimulation. In addition, cyclic AMP phosphodiesterase increases (39) and cyclic AMP-dependent pro

tein kinase decreases (37) have been reported. These effectsall serve to increase the expression of the influence of cyclicGMP relative to that of cyclic AMP on cellular events.

The initiation of lymphocyte proliferation by mitogens isassociated with a rapid Ca2*-dependent increase in cyclic

GMP (7, 9, 28, 50). PHA stimulates these increases through

3 The abbreviations used are: PHA. phytohemagglutinin; PMA, phorbol-12-myristate-13-acetate (also termed 12-O-tetradecanoyl-phorbol-13-acetate);cyclic GMP, cyclic guanosine 3':5'-monophosphate; cyclic AMP, cyclic adenosine 3':5'-monosphosphate; 15-HETE, hydroxyeicosatetraenoic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; NDGA, nordihydroguaiaretic

acid.

150 CANCER RESEARCH VOL. 43

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PMA on Lymphocyte Cyclic Nucleotides

activation of guanylate cyclase with no apparent effects onphosphodiesterase activities (10). We have reported that PMAincreases cyclic GMP levels (12) and activates lymphocyteguanylate cyclase (11 ). We now report that, coincident withthis stimulation, 2 other rapid changes in cyclic nucleotidemetabolism occur: an increase in cyclic GMP phosphodiesterase; and a reduction in adenylate cyclase activity. Inhibitorstudies suggest different mechanisms for these effects of PMA.

MATERIALS AND METHODS

PMA was obtained from Consolidated Midland Corp. (Brewster,N. Y.). Phorbol, phorbol diacetate, phorbol dibutyrate, phorbol diben-

zoate, and phorbol didecanoate were obtained from Sigma ChemicalCo. (St. Louis, Mo.). Methoxyverapamil was a generous gift from A. G.Knoll (Ludwigshafen am Rhein, West Germany). 15-HETE was a gen

erous gift from Dr. J. Martyn Bailey (Georgetown University, Washington, D. C.). Dr. Peter K. Chiang (NIMD, Bethesda, Md.) kindly provided3-deazaadenosine. 3-lsobutyl-1-methylxanthine was from Aldrich

Chemical Co. (Milwaukee, Wis.). Aguasol and all radioactive materialswere obtained from New England Nuclear (Boston, Mass.) and wereused without further purification except where noted below. Highlyspecific antibodies (effective at 1:10,000 dilution) to cyclic AMP andcyclic GMP were prepared in collaboration with Dr. Y. B. Kim accordingto the procedures of Steiner ef al. (55). AG 1-X8 (100 to 200 mesh)was obtained from Bio-Rad Laboratories (Richmond, Calif.), and neutral

aluminum oxide was obtained from E. M. Laboratories (Elmsford,N. Y.). All other chemicals were from Sigma.

Peripheral blood lymphocytes were purified from heparinized venousblood of healthy adults as described (28) and were suspended inHanks' balanced salt solution at a concentration of 10 x 106/ml. Cell

preparations were >98% pure mononuclear cells (>85% T- and B-

lymphocytes by morphological criteria), and platelet contaminationranged from 0 to 0.1% by weight. Cells were equilibrated at 37Â° for

>30 min prior to the addition of PMA or other agents. Phorbol andphorbol diesters were dissolved in dimethylsulfoxide and added in 1-to 2-/il aliquots to 2 to 4 ml cells. The final concentration of dimethyl

sulfoxide did not exceed 0.1% and had no effect on the activitiesmeasured. Lymphocytes were incubated for 10 min with an optimalmitogenic concentration (1 /Â¿g/ml)of PMA unless otherwise stated. Ifcyclic nucleotide levels were to be measured, cold perchloric acid (finalconcentration, 0.5 M) was added, and samples were purified andassayed as described below. If enzyme activities were to be measured,the cells were centrifuged at 800 x g for 2 min, washed, and homogenized, and membrane and soluble (48,000 x g) components wereprepared as described (10). Guanylate cyclase activity was assayedby incubation in duplicate at 37Â°for 6 min in a total volume of 0.2 ml

containing 20 to 50/ig lymphocyte protein, 0.5 mw GTP, 50 mM HEPES â€¢Na (pH 7.6), 0.5 mM phosphocreatine, 3 units of creatine phosphoki-nase, 0.5 mw 3-isobutyl-1-methylxanthine, and either 5 mM CaCI2, 5

mM MgCI2, or 2 mM MnCI2 unless otherwise stated. The reaction wasterminated with 1 ml of cold 0.5 M perchloric acid, and cyclic GMP waspurified as described below.

Adenylate cyclase was assayed only in membrane fractions, sincepreliminary experiments indicated that negligible amounts of this enzyme from control or PMA-treated lymphocytes were soluble after

centrifugation at 48,000 x g. Aliquots of membrane suspensions wereincubated at 37Â°for 10 min in a total volume of 0.1 ml containing 10

to 25 fig of lymphocyte protein, 50 mM HEPES-Na (pH 7.6), 0.5 mMATP, 10 mM MgCI2, 0.5 mw phosphocreatine, creatine phosphokinase(0.02 mg/ml), and 0.5 mM 3-isobutyl-1-methylxanthine. The reaction

was terminated as above.Cyclic nucleotides were purified by a 2-column procedure using

Minutes after PMAChart 1. Human peripheral blood lymphocytes were prepared as described

in "Materials and Methods" and equilibrated in Hanks' balanced salt solution 30x 10e cells/3 ml. at 37Â°for 30 min. PMA was added in 2 /il of dimethyl sulfoxide

at a final concentration of 1 fig/ml. Cellular levels of cyclic nucleotides wereassayed after perchloric acid termination at the indicated times. Guanylatecyclase was measured in the membrane (GCm) and soluble (GCs) fractions,adenylate cyclase (AC) was measured in the membrane, and cyclic GMP phosphodiesterase (G-PDE) was measured in the soluble fraction as described in"Materials and Methods." The Mg2'-dependent guanylate cyclase is plotted;

approximately the same patterns of changes occurred with enzyme measured inthe presence of Ca2* or Mn2*. Isoproterenol- or NaF-stimulated adenylate

cyclase activities were depressed to about the same extent as were the basalactivities shown. Means Â±S.E. of 7 experiments (bars) are given for 1 to 10 min,and means of 2 experiments for the 20 to 60 min points are given as PMA/control ratios. Control activities were: Mg2*-dependent guanylate cyclase, mem

brane, 4.03; guanylate cyclase, soluble: 2.29; basal adenylate cyclase, 29;cyclic GMP phosphodiesterase. 35 pmol/min/mg protein. Control levels of cyclicGMP (cGMP) and cyclic AMP (cAMP) were 0.30 and 20 pmol/mg cellularprotein, respectively. No effects of PMA on cyclic AMP phosphodiesterase wereobserved. Dimethyl sulfoxide at a final concentration of O.f to 0.5% in thepreincubation medium had no effect on any of the activities measured.

Table 1

Effects of PMA added direct/y to broken cell systems

Human peripheral blood lymphocytes were homogenized, and rviembrane and soluble fractions wereprepared as described in "Materials and Methods." PMA was added 10 min before substrate; guanylatecyclase was determined in a 6-min, 37Â°incubation with GTP and the indicated cation by radioimmunoassay

for cyclic GMP as described.

% of control specificactivityMembranePMA

(ng/ml)10

1001000Ca2104

Â±112 Â±109 Â±*9a

1313Mg2

R. G. Coffey and J. W. Madden

Table 2

Effects of PMA preincubation with intact cellsLymphocytes were incubated with or without PMA (1 (ig/ml) in Hanks' balanced salt solution at 37Â°for

10 min for enzyme assays or for 1 min for cellular levels of cyclic nucleotides. Membrane and solublefractions were prepared and enzymes were assayed as described in "Materials and Methods." Specific

activities for enzymes are the means of pmol/min/mg protein. Cellular levels of cyclic nucleotides are givenas pmol/mg protein.

Controls, specific activity PMA/control P'

Guanylate cyclase,membraneCa2*Mg2*Mn2*Guanylate

cyclase,solubleCa2*Mg2*Mn2*Adenylate

cyclase,membraneBasalIsoproterenolNaFPhosphodiesterase,

solubleCyclicGMP (0.1MM)CyclicGMP (1 .0HM)CyclicGMP (1OHM)CyclicGMP (1 00JIM)CyclicAMP (1MM)Cellular

CyclicGMPCellular

Cyclic AMP0.612.867.730.431.604.4529.175.194.912.836.71785431840.3020.2Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±+Â±0.126(13)c0.531.730.100.390.844.010.416.01.26.54554200.032.1(19)(19)(11)(17)(17)(18)(14)(8)(6)(13)(3)(3)(11)(20)(20)1.671.641.752.893.023.040.410.450.581.531.871.911.461.011.711.10Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±0.210.090.110.190.240.190.030.040.070.110.200.260.130.050.120.09


Table 3

Incubation with PMA for extended timesLymphocytes were incubated in Eagle's minimal essential medium containing 4% human AB+ serum in

a 37Â°, 5% COz/95% air incubator for 18 hr. PMA (1 fig/ml) was added at various times; the total hr

exposure to PMA are indicated. Cells were then washed, and enzymes were assayed as described. All dataare mean values of percentage of control

100 x PMA

Control

for duplicate samples in a single experiment. Control specific activities were as follows: Guanylate cyclase,membrane: Mg2*, 0.41; Mn2*, 2.57. Guanylate cyclase, soluble: Mg2*. 1.41; Mn2*. 5.38. Adenylatecyclase, membrane, basal: 12.0. Adenylate cyclase, isoproterenol (10~5 M)+ GTP (10 4 M): 30.3. Phos-

phodiesterase, membrane, 1 fiM cyclic GMP: 35.8: Phosphodiesterase. membrane, 1 fiM cyclic AMP: 27.8.Phosphodiesterase, soluble, 1 fiw cyclic GMP: 25.2. Phosphodiesterase, soluble, 1 UM cyclic AMP: 106.

% of control

Guanylate cyclase

Membrane SolubleAdenylate cyclase

(membrane)

Phosphodiesterases

Membrane Soluble

Cyclic Cyclic Cyclic CyclicTime Mg2* Mn'* Mg2* Mn2* Basal Isoproterenol GMP AMP GMP AMP

2121827311411317717276203165201193139150865952724555995946928265231167185113116100

with Ca2+, Mg2+, or Mn2+. Similarly, the reduction in adenylate

cyclase activity was also independent of the assay conditions(basal, enzyme stimulated by NaF, or enzyme stimulated byisoproterenol plus GTP). Increases in cyclic GMP phosphodi-

esterase were somewhat greater with the intermediate (1 and10 (UM)concentrations of substrate than with higher or lowercyclic GMP concentrations. All of these changes were highlysignificant (p < 0.05) and occurred in preparations of lymphocytes freed of monocytes by glass adherence as well as inpreparations completely depleted of platelets by overnightincubation.

In an attempt to evaluate the stability of the changes inducedby PMA, cells were incubated for varying periods with PMA in4% serum-containing Eagle's minimal essential medium. The

results (Table 3) indicate that by 12 hr the guanylate cyclaseactivities were returning toward control, but the soluble enzymeremained elevated up to 18 hr. Adenylate cyclase remaineddecreased until 18 hr. Membrane cyclic GMP phosphodiester-

ase decreased at 12 hr, while the soluble activity reached apeak much earlier and remained above control at 18 hr. Asignificant decrease in membrane-bound cyclic AMP phospho-

diesterase occurred at 18 hr, but no changes were noted inthe soluble enzyme. These results indicate that many of theearly changes in enzymes caused by PMA are quite persistent.

PMA dose-response effects on the various activities were

examined in an effort to relate the changes to biological effectsof PMA, which exerts peak mitogenic influence on humanperipheral blood lymphocytes at 0.1 to 1 /Â¿g/ml(18, 56). InChart 2, the effects of PMA on most of the activities increasedwith concentration from 0.01 to 10 jug PMA per ml. An exception is cyclic GMP Phosphodiesterase which reached a plateauat 1 /ig PMA per ml.

Analogues of PMA have been synthesized and comparedwith PMA in mitogenic experiments as well as in tumor promotion protocols (2, 17, 35, 64). In 2 experiments,5 PMA and

phorbol dibutyrate were about equipotent in stimulating lymphocyte DMA synthesis. Phorbol diacetate and phorbol dide-

canoate were of intermediate potency; phorbol dibenzoate was

Coo

Oo:

.01 10PMA

5 E. M. Madden, unpublished observations.

Chart 2. Human peripheral blood lymphocytes were incubated in Hanks'

balanced salt solution with varying amounts of PMA for 10 min, and enzymeswere assayed as described in the legend to Chart 1. Means of 2 experiments areshown as PMA/control ratios. Bars, S.E. Control values were: Mg2*-dependent

guanylate cyclase, membrane (GCm), 0.99; guanylate cyclase soluble (GCs).0.81; basal adenylate cyclase WO. 14.4; cyclic GMP Phosphodiesterase (G-PDE), 65 pmol/min/mg protein. cGMP, cyclic GMP.

less effective; and the parent compound, phorbol, was withouteffect. We compared this series of analogues for their effectson the cyclases and cyclic nucleotide levels. The most effectivecompounds for stimulating cyclic GMP levels were PMA andphorbol didecanoate (Table 4). PMA was the most potent forstimulating guanylate cyclase, while phorbol dibutyrate, phorbol dibenzoate, and phorbol didecanoate were also very effective. Phorbol diacetate stimulated only the membrane form ofthe enzyme, and phorbol was without stimulatory effects oncyclic GMP or guanylate cyclase. This profile is similar but notidentical to that for binding to human peripheral blood lymphocytes (52) and for tumor promotion (35, 64). Compounds thatstimulated guanylate cyclase also reduced adenylate cyclaseactivity with the exception of phorbol diacetate and phorbol.The latter actually stimulated adenylate cyclase.

JANUARY 1983 153



R. G. Coffey and J. W. Hadden

Table 4

Effects of phorbol esters on lymphocyte cyclic nucleotide metabolismLymphocytes were incubated in duplicate at 10 x 106/ml of Hanks' balanced salt solution at 37Â°for 30

min. Phorbol or phorbol ester was then added in 2 .ul so that the final concentration was 1.6 /

PMA on Lymphocyte Cyclic Nucleoides

Table 5

Inhibition oÃ-PMA effectsLymphocytes were equilibrated in Hanks' balanced salt solution at 37Â°for 30 min. Inhibitors were then

added for 10 min [ethylene glycol bis (/?-aminoethyl ether)-W,A/.W,N'-tetraacetic acid; methoxyverapamil;15-HETE; NDGA; indomethacin] or 30 min (3-deazaadenosine, puromycin, actinomycin D, cycloheximide),followed by PMA (1 fig/ml) for 10 min. Assays for Mg; '-dnpnndnnt guanylate cyclase, basal adenylate

cyclase, and soluble cyclic GMP (1 Â¡a*)phosphodiesterase were performed as described above.

% ofinhibitionGuanylate

cyclaseInhibitorsEthylene

glycol bis(/J-amino-ethylether)-N,N,N',N'-tetra-acetic

acid, 5mMMethoxyverapamil,10.MM3-Deazaadenosine,10/IM1

5-HETE, 2.5/iM15-HETE. 5/IM15-HETE, 10fiMNDGA,

1MMNDGA,3.MMNDGA,10fiMNDGA,30JIMIndomethacin,

m,Â«MPuromycin,1fig/mlActinomycinD, 0.1/Â»g/mlActinomycinD, 1.0fig/mlCycloheximide,

1 /ig/mlMembrane(5)fi(2)(3)(2)(2)(2)(1)(3)(2)(1)(1)(2)(3)(1)(1)66825434491Â±

11c'dÂ±18"Â±

11d*

3rfÂ±12dÂ±

3"NDe5976Â±11"Â±

14dND(E:66)01500Â±

10Soluble56873512203216314888Â±12*Â±13"Â±

7aÂ±

6Â±9Â±5Â±

6dÂ±5d(E:44)024137Â±

15Adenylate

cyclase828451630382006165191317Â±Â±Â±Â±Â±Â±NOÂ±NDÂ±Â±11152881319Cyclic

GMPphosphodies

terase330761921623473305011Â±Â±Â±Â±Â±Â±Â±NDÂ±9761710883

a Data represent interference by drugs of PMA-induced suppression of activity.b Numbers in parentheses, number of experiments.c Mean Â±S.E. calculated according to

% of inhibition = 100 < 1 -

where C is control, D is drug, P is PMA, and PD is PMA plus drug." Significant inhibition (p < 0.05).9 ND, not done; E, enhancement of PMA stimulation.

DISCUSSION

The present studies demonstrate the capacity of tumor-pro

ducing phorbol diesters, mitogenic substances unrelated to theplant lectins, to cause in human lymphocytes rapid increasesin guanylate cyclase, cyclic GMP levels, and cyclic GMP phosphodiesterase activities. While PHA had no immediate effecton phosphodiesterase activities and stimulated primarily themembrane-bound guanylate cyclase (10), PMA stimulated thesoluble guanylate cyclase much more than the membraneenzyme. This and other differences between PMA and PHA arecompared in Table 6. The initial PMA-induced increase in

cellular cyclic GMP at 1 min was followed by a decline andthen a greater increase which persisted up to 1 hr. One mayspeculate on the relative contributions of the guanylate cy-

clases and phosphodiesterase to account for the changes incellular levels of cyclic GMP, particularly since phosphodiesterase activity plateaued at a maximum level corresponding tothe decline in cyclic GMP at 2 to 5 min, while the second risein the soluble cyclase corresponded to the second rise in cyclicGMP. However, the unknown compartmentalization of cyclicnucleotides, the relative contributions from subpopulations ofmononuclear cells, and the effects of cell disruption on theenzyme activities make such speculations inappropriate.

Cyclic GMP phosphodiesterase localization is poorly understood at present. The activity has been found to be distributedvariously between the membrane and soluble fractions, depending on the techniques of cell disruption (60). Epstein era/.(15) found that both soluble cyclic AMP and cyclic GMPphosphodiesterases increased in human lymphocytes after 24

Table 6Comparison of early effects of PMA with PHAa in human lymphocytes

PHA PMA

Time of maximum increases in cyclic GMP (min)Activation of membrane guanylate cyclaseActivation of soluble guanylate cyclaseActivation of cyclic GMP phosphodiesteraseSuppression of adenylate cyclaseInhibition of guanylate cyclase activation by:

removal of extracellular Ca2*blocking intracellular Ca2* movement

inhibiting phospholipid methyltransferaseinhibiting phospholipase A.inhibiting cyclooxygenaseinhibiting lipoxygenaseinhibiting macromolecular synthesis

5-10

+ +0

1;20

ND0

The PHA experiments were described previously (10).+, small effect; + + , large effect; 0, no effect, ND, not done.

hr exposure to PHA. Our homogenization procedure yields amembrane fraction containing about 60% of the cellular phosphodiesterase acting on cyclic GMP and 20% acting on cyclicAMP. Rapid increases in soluble cyclic GMP phosphodiesterase due to PMA were not related to changes in the membraneenzyme, which decreased significantly only after 2 hr.

Rochette-Egly ef al. also found that PMA (0.1 /Â¿g/ml)induced

a biphasic increase in cyclic GMP levels in human lymphocytes(50) and rat embryo fibroblasts (49). In these cells (48) as wellas mouse epidermal cells (5, 38, 41), PMA has a pronouncedeffect after a lag time of >1 hr to uncouple adenylate cyclasefrom /S-adrenergic and prostaglandin E, receptor activation.This effect may require de novo synthesis of a short-lived"refractoriness protein" (38). On the other hand, Novogrodsky

JANUARY 1983 155



R. G. Coffey and J. W. Madden

et al. (42) recently reported that the inhibition of /S-adrenergic

stimulation of human lymphocyte adenylate cyclase by PMA ismediated by oxygen radicals produced by macrophages present in the incubations. In most of our studies, basal andisoproterenol-stimulated adenylate cyclase activities were depressed to similar degrees at times of up to 1 hr of exposure toPMA. Only after 2 hr (Table 3) did the depression in /8-adre-

nergic activity exceed the depression in basal activity. It therefore appears that 2 changes may occur in lymphocyte adenylate cyclase: one involving a depression at the catalytic sitepossibly reflecting general membrane perturbations; and theother involving an uncoupling of the /8-adrenergic receptor from

the enzyme.Despite the reduced activity of adenylate cyclase, we did not

observe changes in cyclic AMP levels in PMA-treated cells. In

other cell culture systems, decreases in cyclic AMP often (25,49, 63) but not always (57) accompanied large increases incyclic GMP induced by PMA.

While a decrease in cyclic AMP phosphodiesterase activityat the same time as a decrease in adenylate cyclase wouldaccount for our unchanging cyclic AMP levels, such earlychanges in cyclic AMP phosphodiesterase were not observed.Epstein et al. (15) found no change in human lymphocyte cyclicAMP levels despite large decreases in the cyclase and increases in the phosphodiesterase occurring 24 hr after PHA.Further work is required to explain the apparently stable cyclicAMP levels in our experiments.

Our proposal (9, 27, 28) that cyclic GMP represents animportant component of the mitogenic signal in lymphocyteshas met with support from several laboratories (7, 27, 33, 50).Soon after mitogen stimulation of cyclic GMP levels, elevatedactivity of cyclic GMP-dependent protein kinase has been

observed (7, 59). Malignant blood cells are also characterizedby high levels of cyclic GMP (7). It would be premature tointerpret the present data in terms of this proposal because ofthe heterogeneity of the mononuclear cells; only 20% of thecells stimulated by PMA will divide in 72 hr (17). Further studiesusing isolated T-lymphocyte subpopulations are needed. Also,

it is notable that negligible effects of PMA on enzyme activitieswere observed at 0.01 fig/ml, a concentration which inducesapproximately half-maximal lymphocyte proliferation (18, 56).

However, at such suboptimal concentrations, the kinetics ofearly events may differ, and enzyme activities should be measured at later times. Phorbol diacetate (17) and other analogues4

may have optimal mitogenic concentrations >1 /Â¿g/mland mayvary from one experiment to another. Thus, studies are required in which DNA synthesis and enzyme changes are simultaneously measured over a range of phorbol ester concentrations.

Several studies strongly suggest that mitogens act on thelymphocyte cell surface to stimulate a sequence of biochemicalchanges including phospholipid methylation (32) and phospho-lipase activation (32, 47). Arachidonic acid is released (43)and metabolized via the cyclooxygenase pathway to thrombox-anes and prostaglandins or via the lipoxygenase pathways toform various hydroperoxyeicosatetraenoic acids which arethen converted to the corresponding hydroxyeicosatetraenoicacids or other polar metabolites (21, 43). Inhibition of themethylation, phospholipase A2, or lipoxygenase reactions prevents PHA stimulation of guanylate cyclase (10) and DNAsynthesis (10, 32). Several workers have shown that guanylate

cyclase is stimulated by oxidants arising from the above reactions including that of hydroperoxyeicosatetraenoic acids (13,23, 31). We therefore proposed that mitogens might increasecyclic GMP levels indirectly by stimulating in intact cells, viathe pathways described, the production of lipoxygenase metabolites which may then act as proximal stimulants of guanylate cyclase. Craven and DeRubertis (13) suggested a similarpathway to explain Ca2+ stimulation of cyclic GMP levels in

renal cortex.The inhibitor studies summarized in Tables 5 and 6 suggest

that the guanylate cyclase-stimulating action of PMA, like thatof PHA, requires Ca2+. However, the Ca2+ requirement for the

action of PMA to stimulate guanylate cyclase appears to bemet largely by intracellular Ca2*, while that for PHA is met byextracellular or cell surface-associated Ca2*. PMA also actsthrough an intracellular Ca2+ mechanism in stimulating lyso-

zyme release from human neutrophils (54). The effects of PMAmay be mediated by a sequence of Ca2*-dependent phospho

lipid metabolic changes similar to that proposed for PHA inlymphocytes. PMA apparently does not stimulate phospholipidtransmethylation (32) but does activate arachidonic acidrelease from membrane phospholipids (36,41 ) and subsequentmetabolism via cyclooxygenase (6) and lipoxygenase (62).Two relatively specific lipoxygenase inhibitors, NDGA (29) and15-HETE (21, 58), blocked the activation of guanylate cyclase

by PMA as well as by PHA, while the cyclooxygenase inhibitorindomethacin did not prevent activation. These results suggestthat both PHA and PMA stimulate guanylate cyclase throughthe action of a lipoxygenase intermediate. The fact that membrane guanylate cyclase activation by PMA was more effectively inhibited by NDGA and 15-HETE than the soluble enzyme

indicates that different mechanisms may be involved. The sameconsiderations apply to the effect of PMA on cyclic GMPphosphodiesterase and adenylate cyclase. The use of highlyselective inhibitors in the further exploration of the mechanismof action of PMA and other mitogens should be of great help inunderstanding which of the many effects of PMA on cyclicnucleotide metabolism are critical to the induction of proliferation and promotion.

ACKNOWLEDGMENTS

We would like to acknowledge the assistance of Or. Elba M. Madden inperforming lymphocyte cultures and of Christina S. Coffey in conducting cyclicnucleotide assays.

REFERENCES

1. Abb, J., Bayliss, G. J.. and Deinhardt. F. Lymphocyte activation by thetumor-promoting agent 12-O-tetradecanoyl phorbol 13-acetate (TRA). J.Immunol., 122: 1639-1642, 1979.

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1983;43:150-158. Cancer Res Ronald G. Coffey and John W. Hadden Phosphodiesterase and Reduction of Adenylate Cyclase

-Monophosphate′:5′Cyclase and Cyclic Guanosine 3Phorbol Myristate Acetate Stimulation of Lymphocyte Guanylate

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