Biol Cell (1992) 75, 55-59 © Elsevier, Paris
55
Original article
Lymphocyte calmodulin and its participation in the stimulation of T lymphocytes by mitogenic lectins Hiroshi Nakabayashi ~, Hiroshi K o m a d a 2, Toshimichi Yoshida ~, Hideki Takanari ~, Kosaku Izutsu ~" Departments of i Pathology and 2 Microbiology, Mie University School of Medicine, Tsu, Mie 514, Japan (Received 30 September 1991 ; accepted 21 April 1992) Summary - Calmodulin was purified from human tonsillar lymphocytes utilizing calcium-dependent binding of calmodulin to
fluphenazine-Sepharose. The molecular weight and phosphodiesterase activation of the lymphocyte calmodulin were very similar to those of purified bovine brain calmodulin. Trifluoperazine (TFP), a calmodulin inhibitor, suppressed lymphocyte stimulation as assessed by 3H-thymidine incorporation into DNA of lectin-stimulated lymphocytes. TFP had no effect on the early 45Ca2+ uptake induced by mitogenic lectins, although this latter was inhibited by verapamil which also suppressed the 3H-thymidine incorporation. The results are in keeping with the interpretation that the inhibition of T cell stimulation by TFP was not due to suppression of Ca 2÷ uptake, but due to inactivation of Ca2*-calmodulin complex which might be formed subsequent to Ca 2+ entry into the cell. lymphocyte stimulation / calcium ion / calmodulin / trifluoperazine
Introduction
Evidence has accumulated for the pivotal role of calcium ions (Ca 2+) in lymphocyte stimulation: 1) extracellular Ca 2÷ is essential for the mitogenesis [1, 19] ; 2) mitogenic lectins induce early increase both in 45Ca2+ uptake[12, 19] and in cytoplasmic free calcium ion concentration [13, 19, 20, 37, 38]; and 3) Ca2+-ionophore A23187 [23, 24] and ionomycin [20] have a mitogenic potency, while calciumchannel blockers inhibit lectin-induced stimulation [4, 5, 26, 27]. Many cellular events involving Ca 2÷ are regulated through Ca2÷-calmodulin complex. Calmodulin binds Ca 2+ to form this complex when Ca 2+ concentration increases in response to stimulation, and becomes capable of activating many enzymes [9, 17]. Also, in the regulation of lymphocyte stimulation, calmodulin has been suggested to play an important role, mainly based on the finding that trifluoperazine (TFP) inhibits lectin-induced stimulation [2, 34]. This agent has been reported to preferentially bind to Ca2+-calmodulin complex, thereby inhibiting the activity of various calmodulin-sensitive enzymes [33]. T F P also inhibits the activation of calmodulinmediated Na ÷ / H ÷ antiport, one of the two known pathways used for the increase of intracellular p H [29]. The rise in intracellular p H is a nearly universal response o f quiescent cells to growth factor addition [31] including T lymphocyte stimulation with mitogens and antigens [15, 301. In T-lymphocyte stimulation, increase in cytoplasmic free Ca 2+ concentration should be a necessary step for
* Correspondence and reprints
the calmodulin-mediated activations. On the other hand, chlorpromazine, one of the phenothiazine derivatives to which T F P belongs, is reported to be a calcium channel blocker in vascular smooth muscle cells [11]. Whether or not T F P affects entry of Ca 2÷ into stimulated T lymphocytes remains unknown. Elucidation of this problem seems to be inevitable, especially when we assess, using this inhibitor, the role of calmodulin in the response of T lymphocytes to stimulation with mitogens. In the present study calmodulin was purified from human lymphocytes, some of its properties were studied, and the effect of T F P on lectin-induced T cell stimulation was investigated. In addition, Ca 2+ influx was measured in TFP-treated, lectin-stimulated lymphocytes.
Materials and methods
Cell preparation and cultivation Human lymphocytes were isolated from fresh palatine tonsils resected for tonsillar hypertrophy from otherwise healthy children using Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) [27]. The cells were suspended at 106 cell/ml in TC199 medium (Nakarai Chemicals, Ltd, Kyoto) supplemented with 20% calf serum (Nakarai Chemicals, Ltd, Kyoto). An optimal dose of phytohemagglutinin (PHA, 3.3/zg/ml, Difco Laboratories, Detroit, MI) or concanavalin A (ConA, 10/zg/ml, Calbiochem, La JoUa, CA) for DNA synthesis was added with or without verapamil (Eizai, Osaka) or TFP (Yoshitomi, Osaka), and cultured at 37°C in a humidified atmosphere with 5% C02 for 72 h. For the terminal 3 h, 3 /zCi/ml of 3H-thymidine (Amersham, Buckinghamshire, UK) was added, and the cells were harvested on a glass-fiber filter paper using an automated cell harvester (Rikaken Co, Ltd, Nagoya). The radioactivity was counted by a liquid scintillation counter (LKB, RACK BETA, 1219, Turku, Finland) using a dioxane scintillator.
56
H Nakabayashi et al
Preparation of calmodulin from bovine brain Bovine brain calmodulin was purified by the method of Yazawa et al [42] with a slight modification. Briefly, fresh bovine brains were homogenized in 10 mM imidazol-HCI buffer containing 1 mM ethylene glycol bis (2-aminoethylether)-N, N ' -tetraacetic acid (EGTA), pH 6.3. The homogenate was centrifuged at 8 000 g for 30 min, and calmodulin in the supernatant fluid was purified by precipitation with trichloroacetic acid (TCA), DEAEcellulose anion-exchange chromatography ( 0 . 2 - 0 . 5 M, NaCt linear gradient) and column chromatography with Sephadex G-75.
Preparation of cahnodulin from human lymphocytes For the purpose of isolation of calmodulin from human lymphocytes, we utilized fluphenazine affinity chromatography [7, 16]. Lymphocytes (3 10 ~° cells, 15 g in wet weight) prepared from tonsils of 27 children were sonicated in 30 ml of sonication b u f f e r (10 mM 2 - h y d r o x y e t h y l - p i p e r a z i n e - N ' - 2 ethanesulfonic acid ( H E P E S ) - N a O H , pH 7.0, 1 mM 2-mercaptoethanol (2-ME), 1 mM EGTA), and were centrifuged at 70000g for 1 h. The supernatant fraction was made 1.1 mM in CaCI.~ and 0.5 M in NaCI, and adjusted to pH 7.0 with NaOH. The resulting solution was applied to a 12-ml column (1,6 cm x 6.0 cm) of fluphenazine-Sepharose 6B which had been equilibrated with starting buffer (10 mM HEPES, pH 7.0, 1 mM 2-ME, 0.5 M NaCI, 0.1 mM CaCl:). The material which bound to the fluphenazine-Sepharose 6B column was eluted by the addition of I mM EGTA to the starting buffer in place of 0.1 mM CaCIz
Preparation of fluphenazine-Sepharose Five g of epoxy-activated Sepharose 6B (Pharmacia Fine Chemicals, Uppsala, Sweden) was washed with deionized water on a glass filter, and mixed with 0. I M carbonate buffer, pH 11.0/40070 dioxane, containing 500 mg of fluphenazine-malate (Yoshitomi, Osaka). This reaction mixture was gently stirred for 16 h at 37°C. After coupling, the Sepharose mixture was washed on a glass funnel with the reaction solution, 0.1 M borate buffer (pH 8.4) and 0.1 acetate buffer (pH 4.0). To block any excess oxirane groups, the beads were treated in 1 M monoethanolamine at room temperature for 12 h.
pipetted out and the wall of the tube was washed with distilled water by pipetting, and then the oil layer was discarded. The pellet was dissolved in 50/~ 1 of formic acid, and was counted in a dioxane based scintillator. The uptake of 45Ca2 + per cell volume was calculated from the count of 4-SCa2+ and 3H-water.
Results
The yield o f h u m a n l y m p h o c y t e c a l m o d u l i n o b t a i n e d as the final p r o d u c t was 1.76 mg per 1 g o f s a m p l e p r o t e i n . As is shown in figure 1, the l y m p h o c y t e c a l m o d u l i n was nearly h o m o g e n e o u s on S D S - P A G E . C o n s i s t e n t with the result by G r a b et al on canine cerebral c a l m o d u l i n [14], C a : + c o u l d accelerate the e l e c t r o p h o r e t i c m i g r a t i o n o f h u m a n l y m p h o c y t e c a l m o d u l i n : when 1 m M CaCI 2 was present, the a p p a r e n t relative m o l e c u l a r mass ( M r) was 16 K, whereas in the presence o f 1 m M E G T A , the m o bility was lower a n d the a p p a r e n t M r was 17 K (fig 1). The low basal activity o f the c a l m o d u l i n - d e f i c i e n t , cyclic n u c l e o t i d e P D E was not inhibited by 1 m M E G T A or 50 g M T F P (fig 2). W h e n excess c a l m o d u l i n either f r o m bovine b r a i n s or h u m a n l y m p h o c y t e s was a d d e d to this p r e p a r a t i o n , the e n z y m e activity was s t i m u l a t e d a p p r o x i mately to ten-fold. This activation by c a l m o d u l i n was completely a b o l i s h e d by I m M E G T A or 5 0 ~ M T F P . F u t h e r m o r e , the d o s e - d e p e n d e n t curve was d e t e r m i n e d for h u m a n l y m p h o c y t e c a l m o d u I i n and for b o v i n e b r a i n c a l m o d u l i n on P D E activity in the presence o f excess calcium. It was f o u n d that the two curves o v e r l a p p e d a l m o s t exactly (fig 3). T h e role o f c a l m o d u l i n in l y m p h o c y t e s t i m u l a t i o n was a n a l y z e d with the use o f c a l m o d u l i n a n t a g o n i s t T F P . A calcium-channel blocker, verapamil, was used as the reference to T F P . V e r a p a m i l interferes specifically with the ent r a n c e o f C a 2÷ t h r o u g h t h e p l a s m a m e m b r a n e , c u l m i n a t i n g in the inhibition o f m a n y Ca~-+-dependent
(1)(2)
SDS-polyacrylamide gel electrophoresis (SDS-PA GE) Electrophoresis was performed on 20°7o polyacrylamide gels by the Laemmli system [21] in the presence of either l mM CaCI z or l mM EGTA.
i
s
t i t L
Phosphodiesterase activity A partially purified, soluble, calmodulin-deficient phosphodiesterase (PDE) was prepared from bovine brain according to the method of Watterson et al [39]. The assay system for 3', 5' -cyclic nucleotide phosphodiesterase was based on that of Butcher and Sutherland [6], and inorganic phosphate was measured by the method of Murphy et al [25].
68K 45K
,i:~..!
..~
25K
Calcium uptake The uptake of Ca 2+ by l y m p h o c y t e s was d e t e r m i n e d with 45CACI2 (New England Nuclear, Boston, MA) and 3H-water (New England Nuclear, Boston, MA) by the method of centrifugation of cells through an oil layer (dim-butyl phthalate : corn oil = 100:33) as described previously [18, 19]. Briefly, lymphocytes were incubated at 37°C for 30 min in the culture medium supplemented wiht l0 ¢Ci of 45CaCIz (24.5 mCi/mg) or 100 ~Ci ~H-water (100 mCi/mg), l 106 ceils in l ml of the medium were layered on 0.5 ml of oil layer in a polypropylene microcentrifuge tube, and centrifuged at 8 500 rpm for 2 min. The medium was
q
12.5K r
.z~,
• .....
Fig 1. SDS-PAGE of human tonsillar lymphocyte calmodulin in 1 mM CaC1 z (1) or in 1 mM EGTA (2). M, was 16 K in 1 mM CaCI z and 17 K in 1 mM EGTA.
Lymphocyte calmodulin and its role in T cell stimulation
57 r - - ~ mean+SD
T
__0.4 I g
T
• F-.I--j mean:l: S D ~
.~ 03
6
o
: ..':'1 .....j :.....j
Q. o
~3
~. 4
//!! ////
'O
g
Q-
Co='rnMO.I 0.1 Ol EGTAmM 1.0 TFPpM - 5O No C , ( : ~ i n
o
0.1 0.1 0.1 ---
LO -
--
1.0
50
Bo~ne Bran Cal~Jooncj)
-5O
ConA ConA ConA
+
+
+
+
V
T~
v
TFP
Non-stimulated
Fig 5. Effect of verapamil (V) at 33/zM and TFP at l0 ~M on calcium uptake of lymphocytes stimulated with ConA or PHA. Verapamil inhibited the enhancement of Ca -,+ uptake by mitogens, whereas TFP had no effect. The values are expressed as the mean +_ SD of ten experiments.
5,
Human Lymphocyte C,dmoduin
/
t o meon:~ SD
~ 0.1 :...---
0 --4~
10
I000 ng ColmodulJn
I00
Fig 3. Activity of cyclic nucleotide phosphodiesterase in the presence of various amounts of calmodulin from human lymphocytes or a bovine brain. The values are shown as the mean _+ SD of five experiments.
T
. { ConA IOpg/ml
_c --_~
cellular events including lymphocyte stimulation [4, 26, 27]. Effects of both drugs were investigated on 3Hthymidine incorporation into lymphocyte D N A after 72 h of stimulation and on 45Ca2+ uptake by lymphocytes at the initiation of stimulation. Figure 4 shows the doseaction relationship for the inhibitory effect of verapamil and T F P 3H-thymidine incorporation in ConA-stimulated human tonsillar lymphocytes. Thirty-three/zM verapamil or l0 [zM T F P were sufficient to cause almost complete inhibition of the proliferation. These concentrations of the drugs were selected for the measurement of 45Ca2+ uptake. The effect of T F P on 45Ca 2÷ uptake was determined after 30 min of mitogen a n d / o r T F P addition, because the augmentation of Ca 2÷ uptake due to mitogenic lectins was transient occurring within 30 min [19], and because calmodulin antagonists were effective in an early phase of stimulation [3, 8]. Mitogenic doses of P H A and ConA enhanced Ca 2÷ uptake by lymphocytes to about• 1.3- and 1.6- fold, respectively (fig 5). Verapamil inhibited the increase of Ca 2÷ uptake by either of the mitogens to the control level, whereas T F P had no significant effect on the Ca 2÷ uptake (fig 5).
"", Verapomll
,t"
TFP
'o_
Discussion
"".T
!
O
= Non- sllmulaled
0.3
I
Veropomil
3
I0
33
ond Trifluoperozine ( pM i
4. Effects o f verapamil and trifluoperazine on 3H-thymidine i n c o r p o r a t i o n induced by C o n A . V e r a p a m i l and T F P inhibited the stimulation in a dose-dependent manner. The values are expressed as the mean _ SD o f five experiments. Fig
PHA PHA P H A
Colmod.~tloong)
o.4 0.5
=
1/11 //// //// ////
2
0.1 0.1 0.1
Fig 2. Effects of calcium and TFP on the activation of cyclic nucleotide phosphodiesterase by calmodulin. The enzyme activity was stimulated approximately ten-fold by calmodulins. This activation was completely abolished by l mM EGTA or 50/zM TFP. The values are expressed as the mean _+ standard deviation (SD) of five experiments.
7
...-., .i
K
i.ii~
• "'." '1 ...:...i ." :'.7'.1 -. . .. .: '....,,qi
Since calmodulin is a highly conserved protein and found in all the eukaryotes examined so far, it is conceivable that this protein exists in human tonsillar lymphocytes as well and mediates intraceilular signal transduction involving Ca 2+ in lymphocyte stimulation. The present study revealed that the protein of human lymphocyte origin eluted from the fluphenazine affinity column was practically homogeneous as determined by SDS-PAGE. The M r and enzyme-activating capacity of this lymphocyte calmoduiin was equal to purified bovine brain calmodulin. The result of the affinity c h r o m a t o g r a p h y also indicates that,
58
H Nakabayashi et al
in human lymphocytes, calmodulin is the only material that binds to fluphenazine in a Ca 2÷ -dependent manner. An inhibitory effect of the phenothiazine derivatives, chlorpromazine (CPZ) and TFP, on lymphocyte stimulation has already been reported [2, 3, 8, 10, 22]. C P Z had no effect on the binding on ConA to lymphocyte cell surface [10]. It has been reported that calmodulin antagonists were effective in an early phase of stimulation [3, 8], and the drugs did not inhibit the expression or function of the interleukin 2 receptor (IL-2R) seen in the late phase [22], Cheung et al have examined in detail the drug-sensitive period and shown that C P Z and T F P could be added up to 5 h after addition of P H A with inhibitory effect, but later additions were relatively ineffective [8]. This shows that the drug-sensitive period appears to be restricted to an early phase of stimulation. A calcium influx inhibitor, verapamil, inhibits both Ca 2÷ influx and DNA synthesis, indicating that the suppression of DNA synthesis was caused by the inhibition of Ca 2÷ influx. It must be noted, in this connection, that for a significant inhibition of DNA synthesis by verapamil more than 20 h of exposure to this agent is necessary [26, 27]. On the other hand, T F P inhibits D N A synthesis without suppressing the mitogen-induced Ca 2+ uptake. This result indicates that the inhibition of stimulation caused by T F P is due to suppression of the function of Ca2+-calmod.ulin complex inside the cell. This study has shown that the activation of cyclic nucleotide PDE by human lymphocyte calmodulin was completely abolished by 50 g M T P F (fig 2) and this might occur at somewhat lower concentrations. On the other hand, 33 /~M T F P inhibited the 3H-thymidine incorporation into D N A of ConA-stimulated lymphocytes to the nonstimulated control level (fig 4). Therefore, no discrepancy has been found between the biochemical determination and the in vivo experiment. The target of T F P within the cell, however, might be different from the PDE, since Ca 2+-calmodulin can activate many kinds of enzymes [7, 16, 36] and also calmodulin-mediated Na + / H + antiporter [30] which is inhibited by T F P [29]. The activation of Na + / H ÷ antiport appears to be a primary effect of most mitogens that subsequently leads to cell division [31]. Recent reports, however, have questioned the physiological importance of the activation of the antiport in the subsequent induction of T cell proliferation [15]. It is clear that while Ca 2 + is an essential second messenger for T cell stimulation, a rise in the intracellular Ca 2+ concentration per se is not sufficient for the full process of stimulation. Co-stimulation with phorbol esters, or activation of protein kinase C is obligatory [28, 36, 40]. Three isozymes of protein kinase C are present in T lymphocytes [32]. Szamel et al suggested that protein kinase C plays an important role in lymphocyte stimulation, namely a short term activation of protein kinase C is sufficient for synthesis and expression of IL-2R, while a long term activation of the enzyme is necessary for IL-2 synthesis in human lymphocytes [35]. It should be noted here that while higher concentrations of T F P were reported to inhibit this kinase [41], no such effect is expected to be produced in the range used in the present study : Ober and Pardee [29] observed no inhibitory effect of T F P on protein kinase C at 20 tzM, twice the concentration which was mainly used in this study. From these results, it is suggested that activation of both calmodulin and protein kinase C occurs during the mitogen-induced stimulation of lymphocytes, and that they play an important role in the immune cell function.
Acknowledgments We thank Dr Taro Maeda for his kindness in supplying the surgically resected human tonsils. We are also grateful to Mrs Mari Hara for typing the manuscript.
References 1 Abboud CN, Scully SP, Lichtman AH, Brennan JK, Segel GB (1985) The requirements for ionized calcium and magnesium in lymphocyte proliferation. J Cell Physiol 122, 64 - 72 2 Bachvaroff RJ, Miller F, Rapaport FT (1984) The role of calmodulin in the regulation of human lymphocyte activation. Cell hmnunol 85, 135- 153 3 Baker GA, Santalo R, Blumenstein J (1977) Effect of psychotropic agents upon the blastogenic response of human T-lymphocytes. Biol Psychiatry 12, 159- 169 4 Birx DL, Berger M, Fleisher TA (1984) The interference of T cell activation by calcium channel blocking agents. J hnmunol 133, 2904-2909 5 Blitstein-Willinger E, Diamantstein T (1978) Inhibition by isoptin (a calcium antagonist) of the mitogenic stimulation of lymphocytes prior to the S-phase. Immunology 34, 303 - 307 6 Butcher RW, Sutherland EW (1962) Adenosine 3', 5' phosphate in biological materials. 1. Purification and properties of cyclic 3', 5' -nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3', 5' -phosphate in human urine. J Biol Chem 237, 1244- 1250 7 Charbonneau H, Cormier MJ (1979) Purification of plant calmodulin by fluphenazine-sepharose affinity chromatography. Biochem Biophys Res Commun 90, 1039- 1047 8 Cheung RK, Grinstein S, Gelfand EW (1983) Permissive role of calcium in the inhibition of T cell mitogenesis by calmodulin antagonists. J lmmunol 131, 2291- 2295 9 Cheung WY (1970) Cyclic 3', 5' -nucleotide phosphodiesterase: demonstration of an activator. Biochem Biophys Res Commun 38, 535-538 I0 Ferhuson RM, Schmidtke JR, Simmons RL (1975) Concurrent inhibition by chlorpromazine of concanavalin A-induced lymphocyte aggregation and mitogenesis. Nature (Lond) 256, 744 - 745 I l Flaim SF, Brannan MD, Swigart SC, Gleason MM, Muschek LD (1985) Neuroleptic drugs attenuate calcium influx and tension development in rabbit thoracic aorta: Effects of pimozide, penfluridol, chrorpromazine, and haloperidol. Proc Natl Acad Sci USA 82, 1237- 1241 12 Freedman MH (1979) Early biochemical events in lymphocyte activation. I. Investigations on the nature and significance of early calcium fluxes observed in mitogen-induced T and B lymphocytes. Cell lmmunol 44, 290- 313 13 Gardner P (1989) Calcium and T lymphocyte activation. Cell 59, 15-20 14 Grab D J, Berzins K, Cohen RS, Siekevitz P (1979) Presence of calmodulin in postsynaptic densities isolated from canine cerebral cortex. J Biol Chem 254, 8690- 8696 15 Grinstein S, Smith JD, Onizuka R, Cheung RK, Gelfand EW, Benedict S (1988) Activation of Na÷/H ÷ exchange and the expression of cellular proto-oncogenes in mitogenand phorbol ester-treated lymphocytes. J Biol Chem 263, 8658 - 8665 16 Jamieson GA Jr, Vanaman TC (1979) Calcium-dependent affinity chromatography of calmodulin on an immobilized phenothiazine. Biochem Biophys Res Commun 90, 1048- 1056 17 Kakiuchi S, Yamazaki R, Nakazima H (1970) Properties of a heat-stable phosphodiesterase activating factor isolated from brain extract. Studies on cyclic 3', 5' -nucleotide phosphodiesterase II. Proc Jpn Acad 46, 587- 592 18 Komada H, Nakabayashi H, Hara M, Sawada M, Takahashi T, Shiomi T, lzutsu K (1985) Mechanisms of interleukin-
Lymphocyte calmodulin and its role in T cell stimulation
19
20
21 22
23 24 25 26
27
28 29 30 31
2-mediated signaling and role of calcium in T cell mitogenesis. lmmunol Invest 14, 4 3 5 - 4 4 2 Komada H, Nakabayashi H, Nakano H, Takanari H, Takahashi T, lzutsu K (1987) Calcium ion influx during mitogenic stimulation of lymphocytes. Cell Struct Funct 12, 345 - 355 Komada H, Nakabayashi H, Nakano H, Hara M, Yoshida T, Takanari H, Izutsu K (1989) Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin2 or fura-2. Cell Struct Funct 14, 141- 150 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature (Lond) 227, 6 8 0 - 685 Legrue S J, Munn CG (1986) Comparison of the immunosuppressive effects of cyclosporine, lipid-soluble anesthetics, and calmodulin antagonists. Response to exogeneous interleukin 2. Transplantation 42, 6 7 9 - 6 8 5 Luckasen JR, White JG, Kersey JH (1974) Mitogenic properties of a calcium ionophore, A23187. Proc Natl Acad Sci USA 71, 5 0 8 8 - 5090 Maino VC, Green NM, Crumpton MJ (1974) The role of calcium ions in initiating transformation of lymphocytes. Nature (Lond) 251, 3 2 2 - 3 2 7 Murphy J, Riley JP (1962) A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27, 31 - 36 Nakabayashi H, Komada H, lzutzu K (1978) Mechanism of lymphocyte blastogenesis: mitogenic potency of a Ca 2+ ionophore A23187 and suppression of lectin-induced blastogenesis by calcium antagonists (in Japanese). Trans Soc Pathol Jpn 67, 104- 105 Nakabayashi H (1979) The role of calcium ions in lymphocyte stimulation: the effects on blastogenesis of Ca -'+ influx inhibitors, verapamil, nifedipine and quercetin. Mie Med J 29, 91 - 9 8 Nishizuka Y (1986) Studies and perspectives of protein kinase C. Science 233, 305 - 312 Ober SS, Pardee AB (1987) Both protein kinase C and calcium mediate activation of the N a + / H + antiporter in Chinese hamster embryo fibroblasts. J Cell Physiol 132, 3 1 1 - 3 1 7 Rosoff PM, Terres G (1986) Cyclosporine A inhibits Ca 2+ -dependent stimulation of the Na +/H + antiport in human T cells. J Cell Biol 103, 4 5 7 - 4 6 3 Rozengurt E (1986) Early signals in the mitogenic response. Science 234, 161 - 166
59
32 Shearman MS, Berry M, Oda T, Ase K, Kikkawa U, Nishizuka Y (1988) Isolation of protein kinase C subspecies from a preparation of human T lymphocytes. FEBS lett 234, 387 - 391 33 Speaker MG, Sturgill TW, Orlow S J, Chia GH, Pifko-Hirst S, Rosen OM (1980) The effects of trifluoperazine on the macrophage-like cell line, J774. Ann N Y Acad Sci 356, 1 6 2 - 178 34 Stavitsky AB, Dasch JR, Astrachan L (1984) Effects of trifluoperazine, a calmodulin antagonist, on rabbit T- and B-cell responses to mitogens and antigen. Cell Immuno187, 411 - 423 35 Szamel M, Kracht M, Krebs B, Hubner U, Resch K (1990) Activation signals in human lymphocytes : lnterleukin 2 synthesis and expression of high affinity interleukin 2 receptors require differential signalling for the activation of protein kinase C. Cell Immunol 126, 117 - 128 36 Truneh A, Albert F, Goldstein P, Schmitt-Verhulst AM (1985) Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester. Nature 313, 3 1 8 - 3 2 0 37 Tsien RY, Pozzan T, Rink TJ (1982) T-cell mitogens cause early changes in cytoplasmic free Ca -'+ and membrane potential in lynphocytes. Nature (Lond)295, 6 8 - 7 1 38 Tsien RY, Rink TJ, Poenie M (1985) Measurement ofcytosolic free Ca 2+ in individual small cells using fluorescent microscopy with dual excitation wavelengths. Cell Calcium 6, 145- 157 39 Watterson DM, Sharief F, Vanaman TC 0980) The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J Biol Chem 255, 962 - 975 40 Weiss A, lmboden J, Shoback D, Stobo J (1984) Role of T3 surface molecules in human T-cell activation: T3-dependent activation results in an increase in cytoplasmic free calcium. Proc Nail Acad Sci USA 81, 4169-4173 41 Wise BC, Glass DB, Chou CH, Jen, Raynor RL, Katoh N, Shatzman RC, Turner RS, Kibler RF, Kuo JF (1982) Phospholipid-sensitive Ca 2+ -dependent protein kinase from heart. If. Substrate specificity and inhibition by various agents. J Biol Chem 257, 8489- 8495 42 Yazawa M, Sakuma M, Yagi K (1980) Calmodulins from muscles of marine invertebrates, scallop and sea anemone. Comparison with calmodulins from rabbit skeletal muscle and pig brain. J Biochem 87, 1313-1320
55
Original article
Lymphocyte calmodulin and its participation in the stimulation of T lymphocytes by mitogenic lectins Hiroshi Nakabayashi ~, Hiroshi K o m a d a 2, Toshimichi Yoshida ~, Hideki Takanari ~, Kosaku Izutsu ~" Departments of i Pathology and 2 Microbiology, Mie University School of Medicine, Tsu, Mie 514, Japan (Received 30 September 1991 ; accepted 21 April 1992) Summary - Calmodulin was purified from human tonsillar lymphocytes utilizing calcium-dependent binding of calmodulin to
fluphenazine-Sepharose. The molecular weight and phosphodiesterase activation of the lymphocyte calmodulin were very similar to those of purified bovine brain calmodulin. Trifluoperazine (TFP), a calmodulin inhibitor, suppressed lymphocyte stimulation as assessed by 3H-thymidine incorporation into DNA of lectin-stimulated lymphocytes. TFP had no effect on the early 45Ca2+ uptake induced by mitogenic lectins, although this latter was inhibited by verapamil which also suppressed the 3H-thymidine incorporation. The results are in keeping with the interpretation that the inhibition of T cell stimulation by TFP was not due to suppression of Ca 2÷ uptake, but due to inactivation of Ca2*-calmodulin complex which might be formed subsequent to Ca 2+ entry into the cell. lymphocyte stimulation / calcium ion / calmodulin / trifluoperazine
Introduction
Evidence has accumulated for the pivotal role of calcium ions (Ca 2+) in lymphocyte stimulation: 1) extracellular Ca 2÷ is essential for the mitogenesis [1, 19] ; 2) mitogenic lectins induce early increase both in 45Ca2+ uptake[12, 19] and in cytoplasmic free calcium ion concentration [13, 19, 20, 37, 38]; and 3) Ca2+-ionophore A23187 [23, 24] and ionomycin [20] have a mitogenic potency, while calciumchannel blockers inhibit lectin-induced stimulation [4, 5, 26, 27]. Many cellular events involving Ca 2÷ are regulated through Ca2÷-calmodulin complex. Calmodulin binds Ca 2+ to form this complex when Ca 2+ concentration increases in response to stimulation, and becomes capable of activating many enzymes [9, 17]. Also, in the regulation of lymphocyte stimulation, calmodulin has been suggested to play an important role, mainly based on the finding that trifluoperazine (TFP) inhibits lectin-induced stimulation [2, 34]. This agent has been reported to preferentially bind to Ca2+-calmodulin complex, thereby inhibiting the activity of various calmodulin-sensitive enzymes [33]. T F P also inhibits the activation of calmodulinmediated Na ÷ / H ÷ antiport, one of the two known pathways used for the increase of intracellular p H [29]. The rise in intracellular p H is a nearly universal response o f quiescent cells to growth factor addition [31] including T lymphocyte stimulation with mitogens and antigens [15, 301. In T-lymphocyte stimulation, increase in cytoplasmic free Ca 2+ concentration should be a necessary step for
* Correspondence and reprints
the calmodulin-mediated activations. On the other hand, chlorpromazine, one of the phenothiazine derivatives to which T F P belongs, is reported to be a calcium channel blocker in vascular smooth muscle cells [11]. Whether or not T F P affects entry of Ca 2÷ into stimulated T lymphocytes remains unknown. Elucidation of this problem seems to be inevitable, especially when we assess, using this inhibitor, the role of calmodulin in the response of T lymphocytes to stimulation with mitogens. In the present study calmodulin was purified from human lymphocytes, some of its properties were studied, and the effect of T F P on lectin-induced T cell stimulation was investigated. In addition, Ca 2+ influx was measured in TFP-treated, lectin-stimulated lymphocytes.
Materials and methods
Cell preparation and cultivation Human lymphocytes were isolated from fresh palatine tonsils resected for tonsillar hypertrophy from otherwise healthy children using Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) [27]. The cells were suspended at 106 cell/ml in TC199 medium (Nakarai Chemicals, Ltd, Kyoto) supplemented with 20% calf serum (Nakarai Chemicals, Ltd, Kyoto). An optimal dose of phytohemagglutinin (PHA, 3.3/zg/ml, Difco Laboratories, Detroit, MI) or concanavalin A (ConA, 10/zg/ml, Calbiochem, La JoUa, CA) for DNA synthesis was added with or without verapamil (Eizai, Osaka) or TFP (Yoshitomi, Osaka), and cultured at 37°C in a humidified atmosphere with 5% C02 for 72 h. For the terminal 3 h, 3 /zCi/ml of 3H-thymidine (Amersham, Buckinghamshire, UK) was added, and the cells were harvested on a glass-fiber filter paper using an automated cell harvester (Rikaken Co, Ltd, Nagoya). The radioactivity was counted by a liquid scintillation counter (LKB, RACK BETA, 1219, Turku, Finland) using a dioxane scintillator.
56
H Nakabayashi et al
Preparation of calmodulin from bovine brain Bovine brain calmodulin was purified by the method of Yazawa et al [42] with a slight modification. Briefly, fresh bovine brains were homogenized in 10 mM imidazol-HCI buffer containing 1 mM ethylene glycol bis (2-aminoethylether)-N, N ' -tetraacetic acid (EGTA), pH 6.3. The homogenate was centrifuged at 8 000 g for 30 min, and calmodulin in the supernatant fluid was purified by precipitation with trichloroacetic acid (TCA), DEAEcellulose anion-exchange chromatography ( 0 . 2 - 0 . 5 M, NaCt linear gradient) and column chromatography with Sephadex G-75.
Preparation of cahnodulin from human lymphocytes For the purpose of isolation of calmodulin from human lymphocytes, we utilized fluphenazine affinity chromatography [7, 16]. Lymphocytes (3 10 ~° cells, 15 g in wet weight) prepared from tonsils of 27 children were sonicated in 30 ml of sonication b u f f e r (10 mM 2 - h y d r o x y e t h y l - p i p e r a z i n e - N ' - 2 ethanesulfonic acid ( H E P E S ) - N a O H , pH 7.0, 1 mM 2-mercaptoethanol (2-ME), 1 mM EGTA), and were centrifuged at 70000g for 1 h. The supernatant fraction was made 1.1 mM in CaCI.~ and 0.5 M in NaCI, and adjusted to pH 7.0 with NaOH. The resulting solution was applied to a 12-ml column (1,6 cm x 6.0 cm) of fluphenazine-Sepharose 6B which had been equilibrated with starting buffer (10 mM HEPES, pH 7.0, 1 mM 2-ME, 0.5 M NaCI, 0.1 mM CaCl:). The material which bound to the fluphenazine-Sepharose 6B column was eluted by the addition of I mM EGTA to the starting buffer in place of 0.1 mM CaCIz
Preparation of fluphenazine-Sepharose Five g of epoxy-activated Sepharose 6B (Pharmacia Fine Chemicals, Uppsala, Sweden) was washed with deionized water on a glass filter, and mixed with 0. I M carbonate buffer, pH 11.0/40070 dioxane, containing 500 mg of fluphenazine-malate (Yoshitomi, Osaka). This reaction mixture was gently stirred for 16 h at 37°C. After coupling, the Sepharose mixture was washed on a glass funnel with the reaction solution, 0.1 M borate buffer (pH 8.4) and 0.1 acetate buffer (pH 4.0). To block any excess oxirane groups, the beads were treated in 1 M monoethanolamine at room temperature for 12 h.
pipetted out and the wall of the tube was washed with distilled water by pipetting, and then the oil layer was discarded. The pellet was dissolved in 50/~ 1 of formic acid, and was counted in a dioxane based scintillator. The uptake of 45Ca2 + per cell volume was calculated from the count of 4-SCa2+ and 3H-water.
Results
The yield o f h u m a n l y m p h o c y t e c a l m o d u l i n o b t a i n e d as the final p r o d u c t was 1.76 mg per 1 g o f s a m p l e p r o t e i n . As is shown in figure 1, the l y m p h o c y t e c a l m o d u l i n was nearly h o m o g e n e o u s on S D S - P A G E . C o n s i s t e n t with the result by G r a b et al on canine cerebral c a l m o d u l i n [14], C a : + c o u l d accelerate the e l e c t r o p h o r e t i c m i g r a t i o n o f h u m a n l y m p h o c y t e c a l m o d u l i n : when 1 m M CaCI 2 was present, the a p p a r e n t relative m o l e c u l a r mass ( M r) was 16 K, whereas in the presence o f 1 m M E G T A , the m o bility was lower a n d the a p p a r e n t M r was 17 K (fig 1). The low basal activity o f the c a l m o d u l i n - d e f i c i e n t , cyclic n u c l e o t i d e P D E was not inhibited by 1 m M E G T A or 50 g M T F P (fig 2). W h e n excess c a l m o d u l i n either f r o m bovine b r a i n s or h u m a n l y m p h o c y t e s was a d d e d to this p r e p a r a t i o n , the e n z y m e activity was s t i m u l a t e d a p p r o x i mately to ten-fold. This activation by c a l m o d u l i n was completely a b o l i s h e d by I m M E G T A or 5 0 ~ M T F P . F u t h e r m o r e , the d o s e - d e p e n d e n t curve was d e t e r m i n e d for h u m a n l y m p h o c y t e c a l m o d u I i n and for b o v i n e b r a i n c a l m o d u l i n on P D E activity in the presence o f excess calcium. It was f o u n d that the two curves o v e r l a p p e d a l m o s t exactly (fig 3). T h e role o f c a l m o d u l i n in l y m p h o c y t e s t i m u l a t i o n was a n a l y z e d with the use o f c a l m o d u l i n a n t a g o n i s t T F P . A calcium-channel blocker, verapamil, was used as the reference to T F P . V e r a p a m i l interferes specifically with the ent r a n c e o f C a 2÷ t h r o u g h t h e p l a s m a m e m b r a n e , c u l m i n a t i n g in the inhibition o f m a n y Ca~-+-dependent
(1)(2)
SDS-polyacrylamide gel electrophoresis (SDS-PA GE) Electrophoresis was performed on 20°7o polyacrylamide gels by the Laemmli system [21] in the presence of either l mM CaCI z or l mM EGTA.
i
s
t i t L
Phosphodiesterase activity A partially purified, soluble, calmodulin-deficient phosphodiesterase (PDE) was prepared from bovine brain according to the method of Watterson et al [39]. The assay system for 3', 5' -cyclic nucleotide phosphodiesterase was based on that of Butcher and Sutherland [6], and inorganic phosphate was measured by the method of Murphy et al [25].
68K 45K
,i:~..!
..~
25K
Calcium uptake The uptake of Ca 2+ by l y m p h o c y t e s was d e t e r m i n e d with 45CACI2 (New England Nuclear, Boston, MA) and 3H-water (New England Nuclear, Boston, MA) by the method of centrifugation of cells through an oil layer (dim-butyl phthalate : corn oil = 100:33) as described previously [18, 19]. Briefly, lymphocytes were incubated at 37°C for 30 min in the culture medium supplemented wiht l0 ¢Ci of 45CaCIz (24.5 mCi/mg) or 100 ~Ci ~H-water (100 mCi/mg), l 106 ceils in l ml of the medium were layered on 0.5 ml of oil layer in a polypropylene microcentrifuge tube, and centrifuged at 8 500 rpm for 2 min. The medium was
q
12.5K r
.z~,
• .....
Fig 1. SDS-PAGE of human tonsillar lymphocyte calmodulin in 1 mM CaC1 z (1) or in 1 mM EGTA (2). M, was 16 K in 1 mM CaCI z and 17 K in 1 mM EGTA.
Lymphocyte calmodulin and its role in T cell stimulation
57 r - - ~ mean+SD
T
__0.4 I g
T
• F-.I--j mean:l: S D ~
.~ 03
6
o
: ..':'1 .....j :.....j
Q. o
~3
~. 4
//!! ////
'O
g
Q-
Co='rnMO.I 0.1 Ol EGTAmM 1.0 TFPpM - 5O No C , ( : ~ i n
o
0.1 0.1 0.1 ---
LO -
--
1.0
50
Bo~ne Bran Cal~Jooncj)
-5O
ConA ConA ConA
+
+
+
+
V
T~
v
TFP
Non-stimulated
Fig 5. Effect of verapamil (V) at 33/zM and TFP at l0 ~M on calcium uptake of lymphocytes stimulated with ConA or PHA. Verapamil inhibited the enhancement of Ca -,+ uptake by mitogens, whereas TFP had no effect. The values are expressed as the mean +_ SD of ten experiments.
5,
Human Lymphocyte C,dmoduin
/
t o meon:~ SD
~ 0.1 :...---
0 --4~
10
I000 ng ColmodulJn
I00
Fig 3. Activity of cyclic nucleotide phosphodiesterase in the presence of various amounts of calmodulin from human lymphocytes or a bovine brain. The values are shown as the mean _+ SD of five experiments.
T
. { ConA IOpg/ml
_c --_~
cellular events including lymphocyte stimulation [4, 26, 27]. Effects of both drugs were investigated on 3Hthymidine incorporation into lymphocyte D N A after 72 h of stimulation and on 45Ca2+ uptake by lymphocytes at the initiation of stimulation. Figure 4 shows the doseaction relationship for the inhibitory effect of verapamil and T F P 3H-thymidine incorporation in ConA-stimulated human tonsillar lymphocytes. Thirty-three/zM verapamil or l0 [zM T F P were sufficient to cause almost complete inhibition of the proliferation. These concentrations of the drugs were selected for the measurement of 45Ca2+ uptake. The effect of T F P on 45Ca 2÷ uptake was determined after 30 min of mitogen a n d / o r T F P addition, because the augmentation of Ca 2÷ uptake due to mitogenic lectins was transient occurring within 30 min [19], and because calmodulin antagonists were effective in an early phase of stimulation [3, 8]. Mitogenic doses of P H A and ConA enhanced Ca 2÷ uptake by lymphocytes to about• 1.3- and 1.6- fold, respectively (fig 5). Verapamil inhibited the increase of Ca 2÷ uptake by either of the mitogens to the control level, whereas T F P had no significant effect on the Ca 2÷ uptake (fig 5).
"", Verapomll
,t"
TFP
'o_
Discussion
"".T
!
O
= Non- sllmulaled
0.3
I
Veropomil
3
I0
33
ond Trifluoperozine ( pM i
4. Effects o f verapamil and trifluoperazine on 3H-thymidine i n c o r p o r a t i o n induced by C o n A . V e r a p a m i l and T F P inhibited the stimulation in a dose-dependent manner. The values are expressed as the mean _ SD o f five experiments. Fig
PHA PHA P H A
Colmod.~tloong)
o.4 0.5
=
1/11 //// //// ////
2
0.1 0.1 0.1
Fig 2. Effects of calcium and TFP on the activation of cyclic nucleotide phosphodiesterase by calmodulin. The enzyme activity was stimulated approximately ten-fold by calmodulins. This activation was completely abolished by l mM EGTA or 50/zM TFP. The values are expressed as the mean _+ standard deviation (SD) of five experiments.
7
...-., .i
K
i.ii~
• "'." '1 ...:...i ." :'.7'.1 -. . .. .: '....,,qi
Since calmodulin is a highly conserved protein and found in all the eukaryotes examined so far, it is conceivable that this protein exists in human tonsillar lymphocytes as well and mediates intraceilular signal transduction involving Ca 2+ in lymphocyte stimulation. The present study revealed that the protein of human lymphocyte origin eluted from the fluphenazine affinity column was practically homogeneous as determined by SDS-PAGE. The M r and enzyme-activating capacity of this lymphocyte calmoduiin was equal to purified bovine brain calmodulin. The result of the affinity c h r o m a t o g r a p h y also indicates that,
58
H Nakabayashi et al
in human lymphocytes, calmodulin is the only material that binds to fluphenazine in a Ca 2÷ -dependent manner. An inhibitory effect of the phenothiazine derivatives, chlorpromazine (CPZ) and TFP, on lymphocyte stimulation has already been reported [2, 3, 8, 10, 22]. C P Z had no effect on the binding on ConA to lymphocyte cell surface [10]. It has been reported that calmodulin antagonists were effective in an early phase of stimulation [3, 8], and the drugs did not inhibit the expression or function of the interleukin 2 receptor (IL-2R) seen in the late phase [22], Cheung et al have examined in detail the drug-sensitive period and shown that C P Z and T F P could be added up to 5 h after addition of P H A with inhibitory effect, but later additions were relatively ineffective [8]. This shows that the drug-sensitive period appears to be restricted to an early phase of stimulation. A calcium influx inhibitor, verapamil, inhibits both Ca 2÷ influx and DNA synthesis, indicating that the suppression of DNA synthesis was caused by the inhibition of Ca 2÷ influx. It must be noted, in this connection, that for a significant inhibition of DNA synthesis by verapamil more than 20 h of exposure to this agent is necessary [26, 27]. On the other hand, T F P inhibits D N A synthesis without suppressing the mitogen-induced Ca 2+ uptake. This result indicates that the inhibition of stimulation caused by T F P is due to suppression of the function of Ca2+-calmod.ulin complex inside the cell. This study has shown that the activation of cyclic nucleotide PDE by human lymphocyte calmodulin was completely abolished by 50 g M T P F (fig 2) and this might occur at somewhat lower concentrations. On the other hand, 33 /~M T F P inhibited the 3H-thymidine incorporation into D N A of ConA-stimulated lymphocytes to the nonstimulated control level (fig 4). Therefore, no discrepancy has been found between the biochemical determination and the in vivo experiment. The target of T F P within the cell, however, might be different from the PDE, since Ca 2+-calmodulin can activate many kinds of enzymes [7, 16, 36] and also calmodulin-mediated Na + / H + antiporter [30] which is inhibited by T F P [29]. The activation of Na + / H ÷ antiport appears to be a primary effect of most mitogens that subsequently leads to cell division [31]. Recent reports, however, have questioned the physiological importance of the activation of the antiport in the subsequent induction of T cell proliferation [15]. It is clear that while Ca 2 + is an essential second messenger for T cell stimulation, a rise in the intracellular Ca 2+ concentration per se is not sufficient for the full process of stimulation. Co-stimulation with phorbol esters, or activation of protein kinase C is obligatory [28, 36, 40]. Three isozymes of protein kinase C are present in T lymphocytes [32]. Szamel et al suggested that protein kinase C plays an important role in lymphocyte stimulation, namely a short term activation of protein kinase C is sufficient for synthesis and expression of IL-2R, while a long term activation of the enzyme is necessary for IL-2 synthesis in human lymphocytes [35]. It should be noted here that while higher concentrations of T F P were reported to inhibit this kinase [41], no such effect is expected to be produced in the range used in the present study : Ober and Pardee [29] observed no inhibitory effect of T F P on protein kinase C at 20 tzM, twice the concentration which was mainly used in this study. From these results, it is suggested that activation of both calmodulin and protein kinase C occurs during the mitogen-induced stimulation of lymphocytes, and that they play an important role in the immune cell function.
Acknowledgments We thank Dr Taro Maeda for his kindness in supplying the surgically resected human tonsils. We are also grateful to Mrs Mari Hara for typing the manuscript.
References 1 Abboud CN, Scully SP, Lichtman AH, Brennan JK, Segel GB (1985) The requirements for ionized calcium and magnesium in lymphocyte proliferation. J Cell Physiol 122, 64 - 72 2 Bachvaroff RJ, Miller F, Rapaport FT (1984) The role of calmodulin in the regulation of human lymphocyte activation. Cell hmnunol 85, 135- 153 3 Baker GA, Santalo R, Blumenstein J (1977) Effect of psychotropic agents upon the blastogenic response of human T-lymphocytes. Biol Psychiatry 12, 159- 169 4 Birx DL, Berger M, Fleisher TA (1984) The interference of T cell activation by calcium channel blocking agents. J hnmunol 133, 2904-2909 5 Blitstein-Willinger E, Diamantstein T (1978) Inhibition by isoptin (a calcium antagonist) of the mitogenic stimulation of lymphocytes prior to the S-phase. Immunology 34, 303 - 307 6 Butcher RW, Sutherland EW (1962) Adenosine 3', 5' phosphate in biological materials. 1. Purification and properties of cyclic 3', 5' -nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3', 5' -phosphate in human urine. J Biol Chem 237, 1244- 1250 7 Charbonneau H, Cormier MJ (1979) Purification of plant calmodulin by fluphenazine-sepharose affinity chromatography. Biochem Biophys Res Commun 90, 1039- 1047 8 Cheung RK, Grinstein S, Gelfand EW (1983) Permissive role of calcium in the inhibition of T cell mitogenesis by calmodulin antagonists. J lmmunol 131, 2291- 2295 9 Cheung WY (1970) Cyclic 3', 5' -nucleotide phosphodiesterase: demonstration of an activator. Biochem Biophys Res Commun 38, 535-538 I0 Ferhuson RM, Schmidtke JR, Simmons RL (1975) Concurrent inhibition by chlorpromazine of concanavalin A-induced lymphocyte aggregation and mitogenesis. Nature (Lond) 256, 744 - 745 I l Flaim SF, Brannan MD, Swigart SC, Gleason MM, Muschek LD (1985) Neuroleptic drugs attenuate calcium influx and tension development in rabbit thoracic aorta: Effects of pimozide, penfluridol, chrorpromazine, and haloperidol. Proc Natl Acad Sci USA 82, 1237- 1241 12 Freedman MH (1979) Early biochemical events in lymphocyte activation. I. Investigations on the nature and significance of early calcium fluxes observed in mitogen-induced T and B lymphocytes. Cell lmmunol 44, 290- 313 13 Gardner P (1989) Calcium and T lymphocyte activation. Cell 59, 15-20 14 Grab D J, Berzins K, Cohen RS, Siekevitz P (1979) Presence of calmodulin in postsynaptic densities isolated from canine cerebral cortex. J Biol Chem 254, 8690- 8696 15 Grinstein S, Smith JD, Onizuka R, Cheung RK, Gelfand EW, Benedict S (1988) Activation of Na÷/H ÷ exchange and the expression of cellular proto-oncogenes in mitogenand phorbol ester-treated lymphocytes. J Biol Chem 263, 8658 - 8665 16 Jamieson GA Jr, Vanaman TC (1979) Calcium-dependent affinity chromatography of calmodulin on an immobilized phenothiazine. Biochem Biophys Res Commun 90, 1048- 1056 17 Kakiuchi S, Yamazaki R, Nakazima H (1970) Properties of a heat-stable phosphodiesterase activating factor isolated from brain extract. Studies on cyclic 3', 5' -nucleotide phosphodiesterase II. Proc Jpn Acad 46, 587- 592 18 Komada H, Nakabayashi H, Hara M, Sawada M, Takahashi T, Shiomi T, lzutsu K (1985) Mechanisms of interleukin-
Lymphocyte calmodulin and its role in T cell stimulation
19
20
21 22
23 24 25 26
27
28 29 30 31
2-mediated signaling and role of calcium in T cell mitogenesis. lmmunol Invest 14, 4 3 5 - 4 4 2 Komada H, Nakabayashi H, Nakano H, Takanari H, Takahashi T, lzutsu K (1987) Calcium ion influx during mitogenic stimulation of lymphocytes. Cell Struct Funct 12, 345 - 355 Komada H, Nakabayashi H, Nakano H, Hara M, Yoshida T, Takanari H, Izutsu K (1989) Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin2 or fura-2. Cell Struct Funct 14, 141- 150 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature (Lond) 227, 6 8 0 - 685 Legrue S J, Munn CG (1986) Comparison of the immunosuppressive effects of cyclosporine, lipid-soluble anesthetics, and calmodulin antagonists. Response to exogeneous interleukin 2. Transplantation 42, 6 7 9 - 6 8 5 Luckasen JR, White JG, Kersey JH (1974) Mitogenic properties of a calcium ionophore, A23187. Proc Natl Acad Sci USA 71, 5 0 8 8 - 5090 Maino VC, Green NM, Crumpton MJ (1974) The role of calcium ions in initiating transformation of lymphocytes. Nature (Lond) 251, 3 2 2 - 3 2 7 Murphy J, Riley JP (1962) A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27, 31 - 36 Nakabayashi H, Komada H, lzutzu K (1978) Mechanism of lymphocyte blastogenesis: mitogenic potency of a Ca 2+ ionophore A23187 and suppression of lectin-induced blastogenesis by calcium antagonists (in Japanese). Trans Soc Pathol Jpn 67, 104- 105 Nakabayashi H (1979) The role of calcium ions in lymphocyte stimulation: the effects on blastogenesis of Ca -'+ influx inhibitors, verapamil, nifedipine and quercetin. Mie Med J 29, 91 - 9 8 Nishizuka Y (1986) Studies and perspectives of protein kinase C. Science 233, 305 - 312 Ober SS, Pardee AB (1987) Both protein kinase C and calcium mediate activation of the N a + / H + antiporter in Chinese hamster embryo fibroblasts. J Cell Physiol 132, 3 1 1 - 3 1 7 Rosoff PM, Terres G (1986) Cyclosporine A inhibits Ca 2+ -dependent stimulation of the Na +/H + antiport in human T cells. J Cell Biol 103, 4 5 7 - 4 6 3 Rozengurt E (1986) Early signals in the mitogenic response. Science 234, 161 - 166
59
32 Shearman MS, Berry M, Oda T, Ase K, Kikkawa U, Nishizuka Y (1988) Isolation of protein kinase C subspecies from a preparation of human T lymphocytes. FEBS lett 234, 387 - 391 33 Speaker MG, Sturgill TW, Orlow S J, Chia GH, Pifko-Hirst S, Rosen OM (1980) The effects of trifluoperazine on the macrophage-like cell line, J774. Ann N Y Acad Sci 356, 1 6 2 - 178 34 Stavitsky AB, Dasch JR, Astrachan L (1984) Effects of trifluoperazine, a calmodulin antagonist, on rabbit T- and B-cell responses to mitogens and antigen. Cell Immuno187, 411 - 423 35 Szamel M, Kracht M, Krebs B, Hubner U, Resch K (1990) Activation signals in human lymphocytes : lnterleukin 2 synthesis and expression of high affinity interleukin 2 receptors require differential signalling for the activation of protein kinase C. Cell Immunol 126, 117 - 128 36 Truneh A, Albert F, Goldstein P, Schmitt-Verhulst AM (1985) Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester. Nature 313, 3 1 8 - 3 2 0 37 Tsien RY, Pozzan T, Rink TJ (1982) T-cell mitogens cause early changes in cytoplasmic free Ca -'+ and membrane potential in lynphocytes. Nature (Lond)295, 6 8 - 7 1 38 Tsien RY, Rink TJ, Poenie M (1985) Measurement ofcytosolic free Ca 2+ in individual small cells using fluorescent microscopy with dual excitation wavelengths. Cell Calcium 6, 145- 157 39 Watterson DM, Sharief F, Vanaman TC 0980) The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J Biol Chem 255, 962 - 975 40 Weiss A, lmboden J, Shoback D, Stobo J (1984) Role of T3 surface molecules in human T-cell activation: T3-dependent activation results in an increase in cytoplasmic free calcium. Proc Nail Acad Sci USA 81, 4169-4173 41 Wise BC, Glass DB, Chou CH, Jen, Raynor RL, Katoh N, Shatzman RC, Turner RS, Kibler RF, Kuo JF (1982) Phospholipid-sensitive Ca 2+ -dependent protein kinase from heart. If. Substrate specificity and inhibition by various agents. J Biol Chem 257, 8489- 8495 42 Yazawa M, Sakuma M, Yagi K (1980) Calmodulins from muscles of marine invertebrates, scallop and sea anemone. Comparison with calmodulins from rabbit skeletal muscle and pig brain. J Biochem 87, 1313-1320
