AbstractCd2+ was found to mimic effectively, potentiate and antagonize the stimulatory action of Ca2+ on myosin light chain kinase (MLCK) and phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK, or protein kinase C). PL-Ca-PK, however, was slightly less sensitive to Cd2+ regulation than was MLCK. Cd2+ also biphasically regulates (i.e., stimulation followed by inhibition) phosphorylation, in the homogenates of the rat caudal artery, of myosin light chain and other endogenous proteins catalyzed by MLCK and PL-Ca-PK. The activation by Cd2+ of MLCK was inhibited by anticalmodulins (e.g., R-24571), whereas the inhibition by a higher Cd2+ concentration of MLCK and PL-Ca-PK was reversed by thiol agents (e.g., cysteine). The present findings may provide one mechanism underlying the vascular toxicity of Cd2+, a major environmental pollutant

Girard, Peggy R.

Kuo, J.F.

Mazzei, Gonzalo J.

Elsevier - Publisher Connector 

Volume 173, number 1 FEBS 1639 July 1984 
Environmental pollutant Cd2+ biphasically and differentially 
regulates myosin light chain kinase and 
phospholipid/Ca2+ -dependent protein kinase 
Gonzalo J. Mazzei, Peggy R. Girard and J.F. Kuo 
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA 
Received 25 May 1984 
Cd’+ was found to mimic effectively, potentiate and antagonize the stimulatory action of Ca2+ on myosin 
light chain kinase (MLCK) and phospholipid-sensitive Ca ‘+-dependent protein kinase (PL-Ca-PK, or 
protein kinase C). PL-Ca-PK, however, was slightly less sensitive to Cd’+ regulation than was MLCK. 
Cd2+ also biphasically regulates (i.e., stimulation followed by inhibition) phosphorylation, in the 
homogenates of the rat caudal artery, of myosin light chain and other endogenous proteins catalyzed by 
MLCK and PL-Ca-PK. The activation by Cd2+ of MLCK was inhibited by anticalmodulins (e.g., 
R-24571), whereas the inhibition by a higher Cd’+ concentration of MLCK and PL-Ca-PK was reversed 
by thiol agents (e.g., cysteine). The present findings may provide one mechanism underlying the vascular 
toxicity of Cd’+, a major environmental pollutant. 
Myosin right chain Cd2+ Vascular toxicity Protein phosphorylation 
1. INTRODUCTION 
A number of in vivo studies have linked ex- 
posure to environmental low levels of Cd’+ to 
pathophysiological changes in cardiovascular 
tissues without manifestations of generalized, 
overt systemic toxicity of the heavy metal [I ,2]. 
The specific changes include hypertension and 
depressed cardiac excitability. Exposure to high 
levels of Cd2+, however, reportedly produces 
hypotensive or normotensive responses in human 
or experimental animals [l-4]. The acute in vitro 
treatments with Cd2+ have been shown to decrease 
contractile response of cardiac muscle [5,6] and 
aorta [7]. Although several modes of action of 
Cd2+ have been suggested [5-lo], the molecular 
mechanisms underlying the toxic effects of Cd’+ 
on blood pressure remain largely unknown. It has 
been shown that phosphorylation of smooth mus- 
cle myosin P light chain (MLC) by myosin light 
chain kinase (MLCK, a calmodulin-sensitive 
Ca2+-dependent protein kinase) causes an in- 
creased actomyosin ATPase and muscle contrac- 
tion [I 11. More recent evidence indicates that 
phospholipid-sensitive Ca2+-dependent protein 
kinase (PL-Ca-PK, protein kinase C) counteracts 
the stimulatory effects of MLCK by phosphoryla- 
ting MLC at different sites [12]. In view of the 
reported cardiovascular toxicity of Cd2+, investi- 
gations into its ability to substitute for Ca2+ in the 
regulation of these two Ca’+-dependent protein 
kinases seem warranted. We report here that Cd2+ 
biphasically and differentially modulated MLCK 
and PL-Ca-PK and phosphorylation of MLC in 
the homogenate of rat caudal artery catalyzed by 
these two enzymes. 
2. EXPERIMENTAL 
2.1. Materials 
Phosphatidylserine (bovine brain) and histone 
Hl (lysine-rich histone, type III-S) were purchased 
from Sigma, St. Louis; calmodulin was from 
Sciogen, Detroit. 
Published by Elsevier Science Publishers B. V. 
00145793/84/$3.00  1984 Federation of European Biochemical Societies 124 
Volume 173, number 1 FEBS LETTERS July 1984 
2.2. Methods 
MLCK was purified to apparent homogeneity 
from bovine heart as in [13]. PL-Ca-PK was par- 
tially purified from bovine heart through the 
Sephacryl step [ 141. MLCK (0.15 pg) was assayed 
essentially as in [ 131. Briefly, the reaction mixture 
(0.2 ml) contained 50 mM Tris-HCl (pH 7.5), 
10 mM MgClz, 0.05 mM EGTA, 1 mM 2-mercap- 
toethanol, 80 gg cardiac MLC, 1OOpM [Y-~~P]- 
ATP (containing about 1 x lo6 cpm), 0.5 pg cal- 
modulin, and various concentrations of CdS04 
and/or CaClz, as indicated. PL-Ca-PK (5.Opg) 
was assayed as in [14]. Briefly, the reaction mix- 
tures (0.2 ml) contained 50 mM Pipes (pH 6.5), 
10 mM MgC12,0.05 mM EGTA, 1 mM 2-mercap- 
toethanol, 20 pg histone Hl, 5 pM [T-~~P]ATP 
(containing about 1 x lo6 cpm), and various con- 
centrations of CdS04 and/or CaClz, as indicated. 
The reactions for the enzymes were carried out at 
30°C for 5 min. Cd’+ was without effect on 
MLCK and PL-Ca-PK in the absence of calmo- 
dulin and phosphatidylserine, respectively. To 
study the ability of Cd’+ to substitute for Ca2+ in 
the activation of MLCK and PL-Ca-PK and 
phosphorylation of their endogenous ubstrates in 
vascular smooth muscle, rat caudal artery (160 mg) 
was cut into small pieces and homogenized with a 
glass homogenizer in 1 ml of 50 mM Tris-HCl 
(pH 7.5) containing 10% glycerol, 50 mM 2-mer- 
A. MLCK B. PL-Ca-PK 
captoethanol and 2 mM EGTA. The homogenate 
(30rg protein), serving as the source of endo- 
genous protein kinases and their substrates, was 
incubated in 0.2 ml of 50 mM Tris-HCI (pH 7.5) 
containing 10 mM MgC12, 1 mM 2-mercaptoetha- 
nol, 0.05 mM EGTA, 14 pM [T-~~P]ATP (contain- 
ing 2 x 10’ cpm) and various additions as indi- 
cated in fig.2. The reaction was carried out at 30°C 
for 15 s. A short reaction time is essential to 
demonstrate the effects of the activators. Electro- 
phoresis of the phosphoproteins in SDS-polyacryl- 
amide gel and subsequent autoradiography were 
performed as in [15]. 
3. RESULTS AND DISCUSSION 
Phosphorylation of MLC by MLCK has been 
shown to increase actomyosin ATPase activity, a 
mechanism responsible for Ca’+-induced smooth 
muscle contraction [ 111. We found that CdS04 
could effectively substitute for CaClz to stimulate 
nearly maximally MLCK and, moreover, could 
potentiate the stimulatory effect of a suboptimal 
concentration of CaC12 (fig. IA). Unlike CaC12, the 
plateau of the MLCK activity was maintained only 
over a narrow range of CdS04 concentration; the 
enzyme activity was markedly inhibited by high 
concentrations of CdS04 (fig. 1A). The concentra- 
tion-related biphasic effects of CdS04 shown 
CONCENTRATION OF CdS04 (FM) 
Fig. 1. Stimulatory and inhibitory effects of Cd*+ on MLCK and PL-Ca-PK assayed in the presence or absence of Ca”. 
MLCK and PL-Ca-PK was assayed as described in section 2 with various concentrations of CdS04 and/or CaC12. 
125 
Volume 173, number 1 FEBS LETTERS July 1984 
above seem to be in agreement with previous fin- 
dings that exposure to Cd2+ at low levels produces 
hypertension whereas at high Ievels produces 
hypotensive (or normotensive) responses in rats 
[ l-4]_ Our data also could explain in part why high 
Cd’+ concentrations decrease contractile responses 
of the isolated vascular tissue. It should be noted 
here that the concentrations of CaClz and CdS04 
indicated in fig.lA and all other studies reported 
herein were those added to the reaction mixtures 
containing 50 PM EGTA; therefore, they were not 
the actual free Ca2+ and Cd2+ concentrations pre- 
sent in the reaction mixtures. 
As shown in fig. IB, Cd2+ also regulated PL-Ca- 
PK in a manner quite similar to that seen above for 
MLCK; one notable difference was that PL-Ca-PK 
was less sensitive to Cd2+ regulation than was 
MLCK. It has been shown that PL-Ca-PK also 
phosphorylates mooth muscle MLC but at sites 
different from those by MLCK [12], resulting in 
attenuation (about 50~0) of actomyosin ATPase 
activity which is activated when MLC is 
phosphorylated by MLCK [12]. It appears that 
Cd’+ like Ca2+, could differentially modulate, in 
a conkentration-related manner, the vascular reac- 
tivity via phosphoryIation by the two Ca2+-depen- 
dent enzymes which have seemingly opposing 
effects. 
The data shown in fig.1 strongly suggest hat 
Cd2+ could mimic Ca2” in activating MLCK, 
presumably by binding to calmodulin in a manner 
similar to Ca’+. This contention was supported by 
our findings that c~modulin antagonists, such as 
trifluoperazine [15], W-7 [16] and R-24571 [17], in- 
deed inhibited to similar extents MLCK activity 
stimulated either by Ca’+, Cd2+, or both (not 
shown). 
To investigate further the biochemical basis of 
the vascular toxicity of Cd’+, we examined and 
compared the effects of Cd2+ and Ca2+ on 
phosphorylation of endogenous proteins in the 
homogenate of rat caudal artery. Autoradiography 
of the phosphoproteins of such studies indicated 
that, in the presence of ~almodulin, CaCl2 
stimulated phosphorylation of MLC (Mr = 20~) 
and two other proteins having higher Mr of 60000 
and 55000 (fig.2). The identities and the functional 
roles of these higher i% proteins are unknown; the 
presence of similar phosphoproteins in tracheal 
smooth muscle has been reported 1181. In the 
126 
2_OK- 
pg(lO~) - + - + - + - * - + - 
c&q(so*, _ + + - _ _ .“” I - w I 
cd((liwj ---_-i--_--_++ 
c ) __- 1 1 raWW3BOlOOlaO 
Fig.2. Autoradiograph showing effects of Cd’+ and 
Ca2+ on phosphorylation of MLC and other endogenous 
proteins in rat caudal artery. The homogenate, serving 
as the source of the endogenous protein kinases and their 
substrate, was assayed as described in section 2 with 
various additions of CdS04, CaC12, phosphatidylserine 
(PS) and caImodulin (CaM). 
presence of calmodulin, CdS04 at a concentration 
as low as I pM could effectively replace CaCI2 to 
stimulate phosphorylation of the 3 proteins. The 
phosphorylation, however, was inhibited by a high 
concentration (100fiM) of CdS04 (fig.2). 
Although phosphatidylserine appeared to be as ef- 
fective as calmodulin in supporting the 
Ca’+-dependent phosphoryIation of MLC, it was 
less effective than caimodulin in stimulating the 
phosphorylation of the other proteins. Moreover, 
CdS04 was less effective than CaCl2 in supporting 
the phospholipid-sensitive phosphorylation of all 
endogenous proteins. These findings, consistent 
with those made with the purified enzyme shown 
above in fig-l, clearly indicated that Cd’+ could 
regulate the phosphory~ation of MLC and other 
arterial proteins catalyzed by MLCK and PL-Ca- 
PK. 
The interactions of Cd’+ with sulfhydryl groups 
in ceIlular proteins have been suggested as a possi- 
ble mechanism by which it produces more 
Volume 173, number 1 FEBS LETTERS 
Table 1 
Reversal of Cd’+ inhibition of MLCK and PL-Ca-PK by cysteine 
Addition [CdSOd] MLCK (pmol/min) PL-Ca-PK (pmollmin) 
CLIM) 
Basal + CaCl2 Basal + CaCl2 
None (control) 0 1.6 55.0 1.5 44.1 
50 46.3 48.2 4.3 46.2 
500 13.0 2.1 0.9 0.7 
Cysteine (10 mM) 0 1.8 62.7 1.1 46.4 
50 44.1 61.3 3.9 50.3 
500 52.8 47.0 4.8 30.9 
July 1984 
Incubation conditions (in 0.2 ml) were the same as in fig.1, with or without 
cysteine, CdS04 and CaCl2, as indicated. EGTA (5O~M) was present in all 
incubations 
generalized cytotoxicity [5,7,19f. In support of this 
contention is the finding that the Cd’+ inhibition 
of the contractile response of cardiovascular 
tissues is reversed by cysteine [5,7]. Here, we found 
that the inhibition of MLCK and PL-Ca-PK by 
5OOpM CdSO.+ was also effectively reversed by 
10 mM cysteine (table 1). Other thiol agents, such 
as 2-mercaptoethanol (10 mM), dithiothreitol 
(l-5 mM), and 2,3-dimercaptopropanol (0.5-1.0 
mM), were also similarly effective (not shown). 
Despite investigations over the years, the 
molecular mechanisms underlying the presser ef- 
fects of Cd’+ are still unclear. The present evidence 
suggests one mechanism for such effects. We 
found recently that PL-Ca-PK phosphorylates 
troponin I and troponin T from cardiac [20,21] 
and skeletal muscle [21]. It remains to be seen 
whether regulation by Cd’+ of troponin 
phosphorylation by the enzyme is related to the 
cardiotoxicity of the metal, 
ACKNOWLEDGEMENTS 
This work was supported by US Public Health 
Service Grants HL-15696, NS-17608 and 
CA-36777, and a Postdoctoral Fellowship from 
Muscular Dystrophy Association of America 
(G.J.M.). 
REFERENCES 
[II 
PI 
[31 
[41 
VI 
WI 
[71 
PI 
PI 
w 
[111 
1121 
u31 
v41 
WI 
Kopp, S.J., Perry, H.M. jr, Glonek, T., Erlanger, 
M., Perry, E.F., Barany, M. and D’Agrosa, L.S. 
(1980) Am. J. Physiol. 239, H22-H30. 
Kopp, S.J., Gloneck, T., Perry, H.M. jr, Erlanger, 
M. and Perry, E.F. (1982) Science 217, 837-839. 
Friberg, L., Piscator, M., Norberg, G. and 
Kjellstrom, T. (1973) in: Cadmium in the 
Environment, 2nd edn, Chemical Rubber Co., 
Cleveland, OH. 
Kopp, S.J., Fischer, V., Erlanger, M., Perry, E.F. 
and Perry, H.M. jr (1978) Proc. Sot. Exp. Biol. 
Med. 159, 339-345. 
Toda, N. (1973) J. Pharmacol. Exp. Ther. 186, 
60-66. 
Pilati, C.F., Ewing, K.L. and Paradise, N.F. (1982) 
Proc. Sot. Exp. Biol. Med. 169, 480-486. 
Toda, N. (1973) Am. J. Physiol. 225, 350-355. 
Teleman, O., Drakenberg, T., Forsen, S. and 
Thulin, E. (1983) Eur. J. Biochem. 134, 453-457. 
Revis, N. (1978) Life Sci. 22, 479-487. 
Caprino, L., Dolci, N., Togna, G., Villa, P., 
Brucci, R. and Carunchio, V. (1982) Toxicol. Appl. 
Pharmacol. 65, 185-188. 
Adelstein, R.S. and Eisenberg, E. (1980) Annu. 
Rev. Biochem. 49, 921-956. 
Nishikawa, M., Hidaka, H. and Adelstein, R.S. 
(1983) J. Biol. Chem. 258, 14069-14072. 
Walsh, M.P., Vallet, B., Autric, F. and Demaille, 
J.G. (1979) J. Biol. Chem. 254, 12136-12144. 
Wise, B.C., Raynor, R.L. and Kuo, J.F. (1982) J. 
Biol. Chem. 257, 8481-8488. 
Wrenn, R.W., Katoh, N., Schatzman, R.C. and 
Kuo, J.F. (1981) Life Sci. 29, 725-733. 
127 
Volume 173, number i FEBS LETTERS July 1984 
[16] Hidaka, H., Naka, M. and Yamaki, T. (1979) Bio- 
them. Biophys. Res. Commun. 90, 694-699. 
1171 Van Belle, H. (1981) Cell Calcium 2, 483-494. 
1181 Hogaboom, KG., Ember, C.A., Butcher, F.R. and 
Fedan, J.S. (1982) FEBS Lett. 139, 309-312. 
[19] Blum, J.J. (1962) Arch. Biochem. Biophys. 97, 
309-320. 
1201 Katoh, N., Wise, B.C. and Kuo, J.F. (1983) 
Biochem. J. 209, 189-195. 
[21] Mazzei, G.J. and Kuo, J.F. (1984) Biochem. J. 
218, 361-369. 
128 


Environmental pollutant Cd2+ biphasically and differentially regulates myosin light chain kinase and phospholipid/Ca2+-dependent protein kinase 

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Environmental pollutant Cd2+ biphasically and differentially regulates myosin light chain kinase and phospholipid/Ca2+-dependent protein kinase

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