374

Biochimica et Biophysica Acta, 4 9 6 ( 1 9 7 7 ) 3 7 4 - - 3 8 3 © Elsevier/North-Holland Biomedical Press

BBA 28144

THE MITOGENIC EFFECT OF A23187 IN HUMAN PERIPHERAL LYMPHOCYTES

P A M E L A J E N S E N *, L A R R Y W I N G E R , H O W A R D PETER NOWELL

RASMUSSEN

** and

Departments of Biochemistry and Biophysics, Pediatrics, and Pathology, School of Medicine~G3, University of Pennsylvania, Philadelphia, Pa. 19174 (U.S.A.) (Received June 15th, 1976) (Revised manuscript received October 11th, 1976)

Summary The mitogenic action of the divalent ionophore A23187 was confirmed and shown to be very sensitive to changes in extracellular calcium ion concentration. At optimal calcium and ionophore concentrations, an increase in [3H]thymidine incorporation was seen that was similar to that seen after phytohemagglutinin addition. A calcium-dependent stimulation of a-aminoisobutyric acid transport was also seen after A23187 addition. Studies with three inhibitors demonstrate a similarity between proliferation induced by phytohemagglutinin and by A23187. Isoproterenol (10 -4 M) and ouabain (10 -7 M) blocked the effects of phytohemagglutinin and A23187. A drug, D-600, that has been shown to block calcium channels in cardiac muscle, inhibited proliferation induced by either phytohemagglutinin or A23187. This concentration of D600 had no effect on either phytohemagglutinin- or A23187induced aSCa2÷ uptake. Furthermore, the (+) and (--) isomers separated from racemic D600, which have been shown to block sodium and calcium channels respectively in smooth muscle, had equal potency in blocking l y m p h o c y t e proliferation.

Introduction When human peripheral lymphocytes are exposed to phytohemagglutinin or concanavallin A, they undergo a change from quiescent, non-proliferating * Present address: Departments of Internal Medicine and Cell Biology, Yale University School of Medicine, New Haven, Conn. 06510 (U.S.A.) ** To w h o m reprint requests should be sent at present address.

375 cells to mitotically-active ones [1,2]. Prior to mitosis the lymphocytes go through a well-defined series of metabolic changes culminating in an increase in DNA synthesis 48--70 h after the original stimulus [1,2]. It is presently thought that the cells activated by these mitogens are primarily the T-cells which normally represent the largest percentage of this heterogenous lymphocyte population [3]. Recent studies of the mechanism of action of these mitogens have concentrated on the possible role of calcium and cyclic nucleotides as intracellular messengers in the cellular response [4--8]. A role for calcium as a critical component in the initiation of l y m p h o c y t e activation is suggested by a variety of experimental observations. Phytohemagglutinin addition is followed by a p r o m p t increase in radiocalcium uptake [9,10]; and in the absence of extracellular Ca 2÷, the mitogen fails to induce a response [4]. Furthermore, the extent of proliferation is a function of external Ca 2÷ concentration in the range of 0.01--0.06 mM [4]. A more precise definition of the role of calcium in the l y m p h o c y t e may be possible by the use of two new classes of drugs. The first, represented by verapamil and its methoxyl derivative, D600, was originally found to block calcium uptake in cardiac and smooth muscle [11,12]. More recently, in studies with isolated islets of Langerhans, D600 has been shown to block glucoseinduced uptake of calcium and, hence, to inhibit glucose-induced insulin release [13]. These results were obtained with racemic mixtures of (+) verapamil or D600. More recent work with the optical (+) and (--) isomers of these drugs has shown that the (--) isomers have a highly specific ability to block the slow Ca 2÷ channel in cardiac muscle, whereas the (+) isomers block primarily the fast Na ÷ channel in the same tissue [14,15]. These findings raise the hope that the (--) isomers of verapamil and D600 may be highly useful drugs with which to explore the role of external Ca 2+ in the activation of specific cells. The second type of drug found to alter cellular calcium is the divalent cation ionophore, of which A23187 is the best example [16]. A23187 has been shown to increase the permeability of natural membranes to calcium and magnesium ions [16,17]. Addition of A23187 has been shown to induce a calciumdependent increase in cell activity in a wide variety of systems, e.g., fluid and electrolyte secretion in the fly and mammalian salivary glands [18,19], histamine release from the mast cells [20,21], and enzyme release from the exocrine pancreas [22,23]. Preliminary reports of the effect of this ionophore upon peripheral lymphocytes have been published [24,25]. The purpose of the present study was to examine more fully the role of Ca 2÷ in the mitogenic response by the use of the drugs, D600 and A23187. Answers to three specific questions were sought: first, if the concentrations of both external calcium and A23187 are properly controlled, can the ionophore induce a mitogenic response that is quantitatively similar to that seen after optimal doses of phytohemagglutinin; second, is ionophore activation similar to that of phytohemagglutinin with regard to both an early parameter of activation [26] and susceptibility to various inhibitors, specifically ouabain [27] and isoproterenol [28]; and third, does D600 block mitogen-induced calcium uptake and thereby the increase in DNA synthesis?

376

Methods

Lymphocyte preparation L y m p h o c y t e s were prepared in either of two ways. For experiments in which proliferation was the only parameter measured, human blood was drawn into heparinized syringes and mixed with twice its volume of calcium- and magnesium-free Hank's balanced salt solution. Density gradient centrifugation at 20°C for 40 min at 400 × g over Ficoll-Hypaque was then carried out [29]. Granulocytes and most of the red blood cells were pelleted. The interface layer, consisting largely of lymphocytes, was collected with a Pasteur pipet and washed twice with Hank's balanced salt solution. For the calcium and amino acid uptake experiments a more homogenous l y m p h o c y t e preparation was needed and the following modifications of the above procedure were designed. Freshly drawn human blood was defibrinated, mixed with Hank's balanced salt solution containing carbonyl iron (0.5 mg/ml blood), and shaken gently for 30 min at 37°C. Monocytes phagocytized the iron [30] and pelleted during the following Ficoll-Hypaque density gradient centrifugation. After a single wash in Hank's balanced salt solution, the cell pellet was taken up in NH4C1/Tris buffer and incubated at 37°C for 10 min to lyse the remaining red blood cells [31]. Cells were spun down at 20°C. The NH4C1/Tris treatment was repeated. The final cell population consisted of 95% lymphocytes with red blood cells and platelets accounting for most of the 5% contamination. Cells were suspended in 1-ml cultures, containing modified Eagle medium, with a calcium concentration of 1.0 mM unless otherwise specified, and 10% human AB serum.

Proliferation measurements The mitogen phytohemagglutinin-M at a final concentration of 50 ~g/ml, or A23187 {dissolved in ethanol to concentrations listed in the text) was added to lymphocytes (0.5 • 106 cells per ml) at the beginning of the culture period. [3H]Thymidine (0.25 pCi) was added for the final 16 h of the culture period and cells were harvested after a total of 64 h in culture by cold trichloroacetic acid precipitation. After being washed once in trichloroacetic acid, the pellet was dissolved in 2 N NH4OH and counted in toluene-Triton X-100 Scintillation fluid.

45CaZ+ uptake L y m p h o c y t e s were incubated in modified Eagle medium with 1.0 mM Ca ~÷ and 10% human serum at a cell concentration of 1 • 106 per ml. Radiocalcium [4SCa2+] (3 pCi) was added to each culture, followed immediately by addition of mitogen or ionophore. After an incubation of 15--20 min at 37 ° C, cells were washed four times in isotonic saline, pH 7.2. The final cell pellet was dissolved in tissue solubilizer and counted in toluene-methyl cellosolve scintillation fluid.

~-Amino.[~4C] isobutyric acid uptake ~-Amino-[14C]isobutyric acid (0.22 mM; 2 t~Ci) was added to 1-ml cultures containing either 0.5 • 106 or 1 • 106 cells/ml. Immediately after, mitogen was given to half the cultures. Cells were incubated at 37°C for various periods of

377 time before being washed four times with cold isotonic saline, pH 7.2, containing 1 mM aminoisobutyric acid. In one experiment 1.1 mM EGTA was added to the standard modified Eagle medium plus serum incubation medium. Counting of the cell pellet was carried out as described above for [4SCa2÷] uptake.

Materials Hank's balanced salt solution, Ca 2÷ and Mg2÷ free, was obtained from Grand Island Biological Company. Modified Eagle medium with Earle's salts was made up from its individual components, purchased from Grand Island Biological Company. In the high-K + medium, Na ÷ was decreased by a corresponding a m o u n t to maintain osmolarity. Ficoll was from Sigma, and Hypaque (sodium salt, 50% solution) from Winthrop Laboratories. Phytohemagglutinin-M was obtained from Difco Laboratories. Thymidine-[3H]methyl and a-amino[1-~4C]isobutyric acid were obtained from New England Nuclear. CaC12 was purchased from either New England Nuclear or Amersham Searle Corporation. The ionophore A23187 was a gift from the Eli Lilly Company; it was stored at --20°C in ethanol as a 2 mM solution. Ouabain and isoproterenol were purchased from Sigma Chemical Company. D600 and its isomers were a gift from the Knoll Pharmaceutical Company; it was stable for several months as a 10 -2 M aqueous solution stored at --20 ° C. Results

Mitogenic effect of A2318 7 The effect of increasing concentrations of A23187 upon DNA synthesis is shown as a function of medium calcium concentration in Fig. 1. Concentrations of ionophore in the range of 0.5--1.0 pM had only a small effect upon [3H]thymidine incorporation at 2.2 mM calcium or even 5.2 mM calcium (data not shown). Maximal incorporation of thymidine was observed between 2 pM and 4 pM ionophore and the extent of stimulation was a function of the Ca 2÷ concentration. When the Ca 2÷ concentration was 0.2 mM, the addition of 2 pM ionophore had very little effect, but if the Ca 2÷ concentration was raised to 2.2 mM then the same concentration of ionophore stimulated thymidine incorporation to the same extent as an optimal concentration of phytohemagglutinin. If the ionophore concentration was raised to 4 #M, then maximal stimulation of DNA synthesis was observed when the extracellular calcium concentration was between 0.7 and 1.2 mM. When the Ca 2÷ concentration was raised to 2.2 mM, then 4 ~M ionophore still stimulated DNA synthesis, but the degree of stimulation was less than maximal. Concentrations of ionophore 6 pM or greater were increasingly inhibitory at all external calcium concentrations and led to cell death as measured by nonexclusion of Trypan Blue. Effect of A23187 on amino acid transport One of the early effects of phytohemagglutinin upon isolated lymphocytes is an increase in the transport of amino acids; this phenomenon is easily measured by following the uptake of amino[14C]isobutyric acid, a non-metabolizable

378 amino acid analog [32]. As previously shown by Mendelsohn et al. [26], there was a delay of approx. 30 min from the time of phytohemagglutinin addition before any significant increase in aminoisobutyric acid transport was noted (Fig. 2). It has also been shown by Mendelsohn et al. [26], that phytohemagglutinin required calcium in order to stimulate aminoisobutyric acid transport. The A23187 effect also required external calcium (data not shown). Also, the concentrations of A23187 needed to induce a change in aminoisobutyric acid uptake were identical to those needed to induce a rise in thymidine incorporation.

5H-Thymidine DPM x 103

o PHA

• A 23187 t4C-aAI B CPM 4 800

I00 90

0 •

PHA 50,u.g/rnl A23187 1.5 x 10"6M

X

Cont

4400

80

4 000

70

:3600

60

:320 0 2800

50

2400 40

2000

30

1600 1200

20 80(

I0

40( |

0.2

t 0.7

t t.2

t 2.2

[Co*'JmM

i

i

I

I

I

2

3

4

I

I

5 6 Hours

I

I

I

i

7

8

9

I0

Fig. 1, T h e r e s p o n s e of h u m a n p e r i p h e r a l l y m p h o c y t e s to p h y t o h e m a g g l u t i n i n ( P H A ) a n d A 2 3 1 8 7 as a f u n c t i o n o f e x t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n . F r e s h l y isolated h u m a n p e r i p h e r a l l y m p h o c y t e s w e r e s u s p e n d e d in m o d i f i e d Eagle m e d i u m w i t h v a r i o u s a m o u n t s of a d d e d c a l c i u m plus 10% AB s e r u m at 0,5 • 106 cells/ml. P h y t o h e m a g g l u t i n i n ( 5 0 # g / m l , o p e n circles) or A 2 3 1 8 7 at v a r i o u s c o n c e n t r a t i o n s (closed circles) was a d d e d , a n d t h e c u l t u r e s w e r e i n c u b a t e d in a C O 2 / a i r a t m o s p h e r e f o r a t o t a l of 64 h. [ 3 H ] T h y m i d i n e ( 0 . 2 5 # C i / m l ) w a s p r e s e n t for t h e final 16 h of i n c u b a t i o n , and t r i c h l o r o a c e t i c acid insoluble r a d i o a c t i v i t y was d e t e r m i n e d . Points are the m e a n of t r i p l i c a t e c u l t u r e s a n d s t a n d a r d e r r o r was less t h a n or e q u a l to 10% of t h e m e a n . Fig. 2. T h e t i m e c o u r s e of u p t a k e o f a m i n o [ 1 4 C ] i $ o b u t y ric acid (14C-AIB) in c o n t r o l a n d m i t o g e n - t r e a t e d h u m a n p e r i p h e r a l l y m p h o c y t e s . L y m p h o c y t e s (0.5 • 106 eells/ml) w e r e s u s p e n d e d in m o d i f i e d Eagle m e d i u m c o n t a i n i n g 10% AB s e r u m a n d i n c u b a t e d in a 3 7 ° C C O 2 / a l r a t m o s p h e r e o v e r n i g h t . T o b e g i n the e x p e r i m e n t , w n i n o [ 1 4 C ] i s o b u t y r i e acid ( 0 . 2 2 raM, 2 pCi) w a s a d d e d to each t u b e , f o l l o w e d i m m e d i a t e l y b y e i t h e r e t h a n o l (10 ~ l / m l ) , A 2 3 1 8 7 (1.5 • 10 -6 M), or p h y t o h e m a g g l u t i n i n ( P H A ) ( 5 0 # g / m l ) . E t h a n o l a l o n e has n o e f f e c t on a m i n o i s o b u t y r i c acid u p t a k e . I n c u b a t i o n was c o n t i n u e d at 3 7 ° C f o r v a r i o u s p e r i o d s of t i m e . T o s t o p t h e u p t a k e , 5 m l o f cold i s o t o n i c saline, p H 7.2~ w a s a d d e d to each t u b e . Cells w e r e c e n t r i f u g e d at 2°C a n d w a s h e d t h r e e m o r e t i m e s in cold saline. Cell pellets w e r e dissolved in tissue solubilizer and c o u n t e d in a scintillation c o u n t e r .

379 TABLE I THE EFFECTS OF OUABAIN AND HIGH EXTERNAL K + UPON THYMIDINE INCORPORATION A23187- AND PHYTOHEMAGGLUTININ-TREATED HUMAN PERIPHERAL LYMPHOCYTES

IN

C u l t u r e c o n d i t i o n s ate e q u i v a l e n t t o t h o s e d e s c r i b e d in Fig. 1, e x c e p t t h a t 2 . 2 m M Ca 2+ w a s p r e s e n t in all c u l t u r e s . T o m a i n t a i n i s o t o n i c i t y in t h e h i g h - K + b u f f e r , a n e q u i v a l e n t a m o u n t o f s o d i u m w a s d e ~ t e d . P o i n t s are t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s a n d s t a n d a r d e r r o r is i n d i c a t e d . Culture condition

[3H] thymidine incorporation ( d p m / 1 0 6 cells)

C o n t r o l (no a d d i t i o n s ) A23187 A23187 + ouabain A23187 + 40 mM K + A 2 3 1 8 7 + 40 m M K + + o u a b a i n Phytohemagglutinin Phytohemagglutinin + ouabain Phytohemagglutinin + 40 mM K + Phytohemagglutinin + 40 mM K + + ouabain

320 26 1 0 0 108 17 6 0 0 18 5 0 0 43 3 0 0 2 200 48 000 42 500

-+ 20 -+ 5 5 0 0 -+ 2 2 -+ 6 0 0 -+ 3 5 0 0 -+ 1 0 0 0 0 -+ 2 0 0 +- 5 0 0 -+ 5500

Effect of inhibitors upon A23187 action When 10 -7 M ouabain is added to human peripheral lymphocytes it blocks the mitogenic effect of phytohemagglutinin. This inhibitory effect of ouabain can be reversed by raising the K + concentration in the incubation medium [27]. 3H-Thyrnidine DPM x 103

o PHA • D600+ PHA

40:

2(?

rO

I

"1' 0,2

~

0

.

5

15xD s _

I

I

I

1.2

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52

Fig. 3. T h e e f f e c t o f d i f f e r e n t c o n c e n t r a t i o n s o f (+) b 6 0 0 u p o n p h y t o h e m a g g l u t i n i n - s t i m u l a t e d t h y m i d i n e i n c o r p o r a t i o n as a f u n c t i o n o f t h e m e d i u m c a l c i u m c o n c e n t r a t i o n . C u l t u r e c o n d i t i o n s are as d e s c r i b e d in Fig. 1. P h y t o h e m a g g l u t i n i n ( P I t A ) , ( o p e n circles) or p h y t o h e m a g g l u t i n i n a n d v a r y i n g c o n c e n t r a t i o n s o f D 6 0 0 , ( c l o s e d circles) w e r e a d d e d to l y m p h o c y t e s at t h e b e g i n n i n g o f t h e c u l t u r e p e r i o d . P o i n t s are t h e m e a n o f t r i p l i c a t e d e t e r m i n a t i o n s a n d s t a n d a r d e r r o r is i n d i c a t e d .

380 As shown in Table I, our results with the mitogen and ouabain were similar to those previously reported. The effect of A23187 was also completely blocked by 10 -7 M ouabain(Table I). The effect of ouabain on both mitogen- and A23187-induced proliferation was reversed by raising the external K ÷ concentration to 40 mM {Table I). Isoproterenol has also been reported to block the mitogenic effect of phytohemagglutinin. It is thought to act by a stimulating adenylate cyclase. Addition of 10 -4 M isoproterenol produced a 50% inhibition of the phytohemagglutinin response and a 60% inhibition of the A23187 response (data not shown).

Effect of D600 upon phytohemagglutinin and A23187 action Increasing concentrations of (-+) D600 caused a progressively greater inhibition of the DNA synthesis induced by mitogen (Fig. 3). In the experiment shown in Fig. 3, addition of 5 • 10 -s M D600 resulted in complete inhibition of [3H]thymidine incorporation. However, the extent of inhibition produced by D600 at any given concentration was variable from donor to donor. Contrary to results found in other systems [11,13], the inhibition caused by D600 was not reversed by raising the external calcium concentration. The results shown in Table II substantiated the independence of D600 inhibition from the influ-

TABLE II THE EFFECT OF D-600 UPON UPTAKE AND [3H]THYMIDINE INCORPORATION IN HUMAN PERIPHERAL LYMPHOCYTES ACTIVATED BY PHYTOHEMAGGLUTININ OR A23187 A single P r e p a r a t i o n o f l y m p h o c y t e s w a s a s s a y e d f o r t h e e f f e c t o f D 6 0 0 o n b o t h p h y t o h e m a g g l u t i n i n i n d u c e d [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n a n d [45Ca2+] u p t a k e in t h i s r e p r e s e n t a t i v e e x p e r i m e n t . L y m p h o c y t e s (2 • 105 c e l l s / m l in m o d i f i e d Eagle m e d i u m a n d 10% AB s e r u m ) w e r e t r e a t e d w i t h D 6 0 0 (5 • 10 - s M), p h y t o h e m a g g l u t i n i n ( 5 0 p g / m l ) , or b o t h a g e n t s . H a l f t h e c u l t u r e s w e r e given t r a c e r 4 5 C a 2+ oneh a l f h o u r a f t e r a d m i n i s t r a t i o n o f m i t o g e n a n d / o r D 6 0 0 . A f t e r 15 m o r e rain at 3 7 ° C , cells w e r e w a s h e d f o u r t i m e s in cold i s o t o n i c N a C l , p H 7.2. Cell pellets w e r e d i s s o l v e d in t i s s u e s o l u b i l i z e r a n d c o u n t e d . T h e o t h e r c u l t u r e s w e r e i n c u b a t e d f o r a t o t a l of 64 h, w i t h [ 3 H ] t h y m i d i n e ( 0 . 2 5 p C i ] m l ) p r e s e n t f o r t h e final 16 h. T r i c h l o r o a c e t i c acid i n s o l u b l e r a d i o a c t i v i t y w a s d e t e r m i n e d b y d i s s o l v i n g p e l l e t s in tissue s o l u b i l i z e r a n d c o u n t i n g . Also, t h e e f f e c t o f t w o d i f f e r e n t c o n c e n t r a t i o n s o f (+-) D 6 0 0 (2 a n d 5 • 10 -5 M) w a s e x a m i n e d w i t h r e s p e c t to 4 S C a 2+ u p t a k e a n d [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n in t h e p r e s e n c e a n d a b s e n c e o f 2 g M A 2 3 1 8 7 . E x p e r i m e n t a l c o n d i t i o n s w e r e t h e s a m e as d e s c r i b e d a b o v e e x c e p t t h a t A 2 3 1 8 7 w a s t h e a d d e d m i t o g e n a n d l y m p h o c y t e s w e r e i n c u b a t e d at 0.5 • 105 cells/ml. Culture conditions

Calcium uptake cpm/15 rain

Thymidine incorporation d p m / 1 0 ° cells

Control (no additions) Ph y t o h e m a g g l u t i n i n Phytohemagglutinin + D 6 0 0 (5 • 10 -5 M) D 6 0 0 (5 • 10 - s M)

86 325

1 300 30 5 0 0

403 171

12 1 0 0 260

c p m / 2 0 rain

d p m / l O 6 cells

110 776 653 667 54 43

1 000 29 1 0 0 17 0 0 0 4 I00 390 240

Control (no additions) A23187 (2 uM) A23187 (2 pM) + D600 (2 • 10 -s M) A23187 (2 ~M) + D500 (5 - 10 -S M) D600 (2 - I 0 -S M) D600 (5 • I 0-S M)

± 200 t 3300 ± 3300 ± 400 -+ 10 ~ 10

381

TABLE III A COMPARISON OF THE EFFECT OF THREE DIFFERENT CONCENTRATIONS O F (+-) D 6 0 0 , (+) D 6 0 0 , A N D (--) D 6 0 0 U P O N A 2 3 1 8 7 - A N D P H Y T O H E M A G G L U T I N I N [3H]THYMIDINE INCORPORATION IN HUMAN PERIPHERAL LYMPHOCYTES C u l t u r e c o n d i t i o n s w e r e as d e s c r i b e d i n F i g . 1 , e x c e p t t h a t p h y t o h e m a g g l u t i n i n o r A 2 3 1 8 7 a n d / o r D 6 0 0 w e r e a d d e d a t t h e b e g i n n i n g o f t h e c u l t u r e p e r i o d t o q u a d r u p l i c a t e t u b e s . S t a n d a r d e r r o r s are i n d i c a t e d . Drug addition

Thymidine incorporation ( d p m / 1 0 6 cells) A23187

Phytohemagglutinin

None

66 000 + 2800

102 000 + 3200

Racemic D600 1 - 1 0 -S M 2 . 5 • 1 0 -5 M 5 • 10 -s M

59 0 0 0 + 2 1 0 0 1 3 0 0 0 +- 1 0 0 0 3 000 + 900

87 000 + 2100 53000 + 4000 25 000 + 1600

(+) D 6 0 0 1 • 10 -s M 2 . 5 • 1 0 -5 M 5 ' 1 0 -5 M

60 000 + 2100 16000 + 1100 3000-+ 700

7 4 0 0 0 -+ 4 2 0 0 37000 + 2100 23000 + 1500

53 0 0 0 + 1 4 0 0 1 3 1 0 0 -+ 1 1 0 0 2000 + 600

72 000 + 4500 3 4 0 0 0 +- 2 6 0 0 1 8 0 0 0 -+ 1 4 0 0

(--) D 6 0 0 1 • 10 -s 2.5 - 10 -s M 5 • 1 0 -5 M

ence of calcium. As other investigators have shown that phytohemagglutinin causes an increase in the uptake of radiocalcium into lymphocytes [9,10], we investigated the effect of D600 in the same cell preparation on both mitogeninduced radiocalcium uptake and [3H]thymidine incorporation. Even though the addition of 5 • 10 -s M D600 caused a 66% inhibition of mitogen-induced [3H]thymidine incorporation it had no effect on mitogen-stimulated radiocalcium uptake (Table II). Also, addition of D600 at a concentration of 2 • 10 -s M caused a 40% inhibition, and at 5 • 10 -s M an 85% inhibition of A23187-induced thymidine uptake (Table II), but neither concentration had an effect on the A23187-induced increase in radiocalcium uptake (Table II). The inhibitory effect of D600 was reversible. If the cells were exposed to 2 • 10 -5 M D600 for 16 h, and then washed and reincubated in fresh media, their responses to both phytohemagglutinin and to A23187 were comparable to control cells incubated, washed and reincubated in the same fashion. In order to further characterize the inhibitory actions of D600 on the l y m p h o c y t e , the effects of the (+) and (--) isomers of D600 upon both the mitogen- and A23187-mediated increases in [3H]thymidine incorporation were examined. The results obtained with phytohemagglutinin- and A23187-treated lymphocytes are shown in Table III. There was no significant difference in the effectiveness of the two optical isomers in blocking either phytohemagglutinin or A23187 action. Discussion The present results add additional evidence in support of the concept that an increase in [Ca 2÷] within some intracellular compartment is an early event in

382 the initiation of proliferation in the peripheral lymphocyte. Our data confirm the fact that A23187 can act as a non-specific mitogen in a population of mixed human peripheral lymphocytes [24,25,33]. They show that, under appropriate conditions of extracellular calcium and ionophore concentration, the degree of stimulation of DNA synthesis is similar to that seen with maximally effective concentrations of phytohemagglutinin. They also show, as previously reported [33], that the mitogenic effect of A23187 is preceded by an A23187-dependent stimulation of calcium uptake (Table II) and that its effect requires the presence of external calcium (Fig. 1) [24,25]. It is important to note that A23187 is not entirely specific for Ca 2÷. It has been shown to increase the permeability of cell and mitochondrial membranes to Mg2÷ [16,17]. Nonetheless, in the present study, when Mg2÷ was present in the incubation medium there was no stimulation of DNA synthesis if Ca 2÷ was removed. Furthermore, Lukasen et al. [24], have shown that removal of Mg 2÷ from the medium results in only a 25% inhibition of the ionophore-induced stimulation of DNA synthesis if Ca 2÷ is present. Our present data (Fig. 2, Table I) extend the evidence showing that the consequences of A23187 action are similar to those following phytohemagglutinin. Both require external calcium for activation (Fig. 1) and both cause an early increase in radiocalcium uptake (Table II, and refs. 9,10,24,25,33). Both stimulate transport of aminoisobutyric acid (ref. 26 and Fig. 2). Phytohemagglutinin also causes a rapid increase in the transport of 3-O-methyl-glucose into lymphocytes [34], and the recent results of Reeves [35] demonstrate that A23187 can increase transport of 3-O-methyl-glucose in a Ca2÷-dependent manner in rat thymocytes. It seems likely that these two latter changes in transport processes are secondary to a rise in intracellular Ca 2÷ and are not consequences of the initial mitogen-membrane interactions. The very close similarity of the A23187 and phytohemagglutinin-induced responses is further supported by the fact that three different classes of inhibitors, ouabain, isoproterenol, and D600, block both types of response (Tables I, II and III). At present the mechanism by which any of these three inhibitors acts is not known, so it is not fruitful to discuss this similarity further. The results obtained with D600 were unexpected. It is reasonable to conclude that D600 is not acting on these cells by blocking a calcium channel in the plasma membrane on the basis of the following facts. The (--) and (+) optical isomers of D600 have similar inhibitory potencies (Table III); D600 does not block phytohemagglutinin-induced calcium uptake (Table II); the inhibition of the phytohemagglutinin response by D600 is not reversed by increasing external [Ca 2÷] (Fig. 3); and D600 blocks A23187-induced mitosis (Tables II and III) as effectively as it does the phytohemagglutinin response. The discovery of this inhibitory effect of D600 may eventually prove of interest and may provide an additional tool to explore the sequence of events involved in activation of these cells to mitosis. However, of more immediate practical importance is the fact that D600 and/or Verapamil are being used by many investigators to explore the possible role of extracellular calcium in cellular response. Our data show that not all of the effects of D600 can be explained by the mechanism worked out in cardiac and smooth muscle [14,15], and that a very complete analysis of its effects, not only on the particular cellular

383

response but on calcium transport, must be carried out before it can be concluded that a specific inhibition of calcium uptake is the basis of its action in a particular cell type. Acknowledgements This work was supported by U.S. Public Health Service Grants CA 12779, CA 15822 and AM 09650. References 1 Nowell, P.C. (1960) Cancer Res. 20,462--466 2 Naspitz, Ch.K. and Richter, M. (1968) in Progress in Allergy (Kallos, P. and Waksman, B.H., eds.), Vol. 12, pp. 1--85, S. Karger, Basel 3 Davies, A.J.S., Festenstein, H., Leuchars, E., Wallis, V.J. and Doenhoff, M.J. (1968) Lancet 1,183-184 4 Whitney, R.B. and Sutherland, R.M. (1972) J. Cell Physiol. 80, 329--337 5 Smith, J.W., Steiner, A.L., Newberry, Jr., W.M. and Parker, C.W. (1971) J. Clin. Invest. 50, 432-441 6 Hadden, J.W., Hadden, E.M., Haddox, M.K. and Goldberg, N.D. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3 024--3027 7 Krishnaro, R. and Talwar, G.P. (1973) J. Immunol. 111, 1010--1017 8 Wedner, H.J., Dankner° R. and Parker, C.W. (1975) J. Imm unol . 115, 1682--1687 9 Whitney, R.B. and Sutherland, R.M. (1973) J. Cell Physiol. 82, 9--20 10 Allwood, G., Asherson, G.L., Davey, M.J. and Goodford, P.S. (1971) I m m u n o l o g y 2 1 , 5 0 9 - - 5 1 6 11 Fleckenstein, A. (1972) in Ca and the Heart (Harris, P. and Opie, L., eds.), pp. 135--188, Academic Press, New York 12 Kroeger, E.A., Marshall, J.M. and Bianchi, C.P. (1975) J. Pharmacol. Exp. Ther. 1 9 3 , 3 0 9 - - 3 1 6 13 Malaisse, W.J., Pipeleers, D . G , Malalsse-Lagae, F. and Orci, L. (1973) Diabetologia 9, 80, Abstract 14 Bayer, R., Kaufman, R. and Mannhold, R. (1975) Arch. Pharmacol. 290, 69--80 15 Bayer, R., Kalusche, D., Kaufman, R. and Mannhold, R. (1975) Naunyn-Schmiedeberg's Arch. Pharmacol. 290, 81--87 16 Reed, P.W. and Lardy, H.A. (1972) J. Biol. Chem. 242, 6970--6977 17 Reed, P.W. and Lardy, H.A. (1972) in The Role of Membranes in Metabolic Regulation (Mehlman, M.A. and Hanson, R.W., eds.) pp. 111--118, Academic Press, New York 18 Prince, W.T., Rasmussen, H. and Berridge, M.J. (1973) Biochim. Biophys. Acta 329, 98--107 19 Selinger, Z., Eimerl, S. and Schramm, M. (1974) Proc. Natl. Acad. Sci. U.S.A. 7 1 , 1 2 8 - - 1 3 1 20 Foreman, J.G., Mongar, J.L. and Gompertz, B.D. (1973) Nature 245, 249--251 21 Cochrane, D.E. and Douglas, W.W. (1974) Proc. Natl. Acad. Sci. U.S. 7 1 , 4 0 8 - - 4 1 2 22 Williams, J.A. and Lee, M. (1974) Biochem. Biophys. Res. Commun. 60, 542--548 23 Eimerl, S.° Savion, N., Heichal, O. and Selinger, Z. (1974) J. Biol. Chem. 249, 3991--3993 24 Luckassen, J.R., White, J.G. and Kersy, J.H. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4 0 8 8 - - 5 0 9 0 25 Malno, V.C., Green, N.M. and Crumpton, M.J. (1974) Nature 2 5 1 , 3 2 4 - - 3 2 7 26 Mendelsohn, J., Skinner, A. and Komfield, S. (1971) J. Clin. Invest. 50, 818--826 27 Quastel, M.R. and Kaplan, J.G. (1968) Nature 2 1 9 , 1 9 8 - - 2 0 0 28 Smith, J.W., Steiner, A.L. and Parker, C.W. (1971) J. Clin. Invest. 5 0 , 4 4 2 - - 4 4 8 29 Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21, Supp. 97, 31--50 30 Zucker-Franklin, D. (1974) J. Immunol. 112, 234--240 31 Boyle, W. (1968) Transplantation 6 , 7 6 1 - - 7 6 4 32 Noall, M.W., Riggs, T.R., Walker, L.M. and Christensen, H.R. (1957) Science 126, 1002--1005 33 Hovi, T., Allison, A.C. and Williams, S.C. (1976) Exptl. CeU Res. 97, 92--100 34 Peters, J.H. and Hausen, P. (1971) Eur. J. Biochem. 19, 509--513 35 Reeves, J.P. (1975) J. Biol. Chem. 250, 9428--9433