Ac. Passaquin et al., CALCIUM INFLUX INHIBITION BY STEROIDS AND ANALOGS IN C2C12 SKELETAL-MUSCLE CELLS, British Journal of Pharmacology, 124(8), 1998, pp. 1751-1759
1 Glucocorticoids, namely alpha-methylprednisolone (PDN) and deflazaco
rt, are the only drugs reported to have a beneficial effect on the deg
enerative course of Duchenne muscular dystrophy (DMD). Increased cytos
olic calcium concentrations ([Ca2+](c)) have been implicated as one of
the pathological events responsible for the degeneration of dystrophi
c skeletal muscles. In previous studies, we have demonstrated that PDN
treatment of both normal and dystrophic murine skeletal muscle cells
was able to normalize elevated [Ca2+](c) and improved myogenesis. Here
we have investigated the mechanism underlying the effects of glucocor
ticoids on cellular Ca2+ influx into C2C12 skeletal muscle cells. 2 Lo
ng-term incubation of C2C12 myocytes with PDN was necessary to observe
a reduction of Ca-45(2+) influx. PDN was most effective in inhibiting
Ca-45(2+) uptake when added for 4 days (at the time of fusion of myob
lasts into myotubes) and to a lesser extent, when added after fusion.
It was ineffective when added to C2C12 cells at the myoblast stage. Sh
ort PDN incubation times, at the time of fusion were insufficient to e
licit a response. 3 Several steroids were tested for their ability to
inhibit Ca-45(2+) influx in C2C12 myocytes. All four glucocorticoids e
xamined were able to reduce Ca2+ influx, dexamethasone being the most
potent (IC50 3.14+/-0.34x10(-8) M). Mineralocorticoids (aldosterone an
d 11-deoxycorticosterone) were also able to reduce Ca2+ influx. 4 The
vitamin E-derived lazaroid U-83836E and the glucocorticoid-derived laz
aroid U-74389G also elicited a decrease in Ca2+ influx, but higher con
centrations were necessary. Because both glucocorticoids and lazaroids
display antioxidant properties, but U-83836E is devoid of glucocortic
oid activity, the reduction in Ca2+ influx was suspected to be trigger
ed via an antioxidant mechanism. 5 To test this hypothesis, we assesse
d the action of several antioxidants, such as vitamin E, vitamin C, 2-
tert.-butyl-4-methoxyphenol (BHA), 2,6-di-tert.-butyl-4-methyl-phenol
(BHT) and nordihydroguaiaretic acid (NDGA), on Ca-45(2+) influx. None
of these agents had an effect on Ca-45(2+) influx. In addition, severa
l oxidants were tested (either acutely or chronically) for their abili
ty to elicit Ca-45(2+) influx in C2C12 myocytes and were found to be i
nactive. 6 The involvement of the glucocorticoid receptor on the modul
ation of Ca2+ influx was investigated. The glucocorticoid receptor ant
agonist mifepristone (code name RU38486, 10(-6) M) caused a shift of t
wo orders of magnitude of the PDN response. However, neither actinomyc
in D nor cycloheximide affected the response to PDN. 7 Results with th
e phospholipase A(2) inhibitor, manoalide, suggest that glucocorticoid
-induced protein synthesis (e.g. enhanced stimulation of lipocortin) d
oes not play a role in the reduction of calcium influx. 8 Our results
suggest that steroids elicit a decrease in calcium influx in C2C12 ske
letal muscle cells. This decrease is not due to an antioxidant mechani
sm or to a mechanism which requires gene expression. Since mineralocor
ticoids and U-83836E also had similar effects, the mechanism could bel
ong to the nongenomic effects of corticoids (e.g. membrane stabilizati
on). The beneficial effect of glucocorticoids in DMD could be attribut
ed to a reduction of the pathological increase in Ca2+ influx via an e
ffect on the sarcolemma.