Sb. Churn et al., BILIRUBIN INDUCES A CALCIUM-DEPENDENT INHIBITION OF MULTIFUNCTIONAL CA2+ CALMODULIN-DEPENDENT KINASE-II ACTIVITY IN-VITRO/, Pediatric research, 38(6), 1995, pp. 949-954
Excessive bilirubin levels in newborn infants result in long term neur
ologic deficits that remain after bilirubin levels return to normal. M
uch of the observed neurologic deficits can be attributed to bilirubin
-induced, delayed neuronal cell death. Inhibition of calcium/calmoduli
n-dependent kinase II (CaM kinase II1) activity that precedes cell dea
th is observed in conditions such as seizure activity, stroke, and glu
tamate excitotoxicity. Because neonatal bilirubin exposure results in
neuronal loss in developing brain systems, we tested whether bilirubin
exposure would induce an immediate inhibition of CaM kinase II activi
ty, in vitro. P-81 filtration assay of basal and calcium-stimulated ki
nase activity was performed under standard kinase assay conditions. Bi
lirubin and/or albumin was added to the reaction vessels to determine
the effect of these agents on kinase activity. Bilirubin exposure resu
lted in a concentration-dependent inhibition of CaM kinase II activity
(IC50 = 16.78 mu M). At concentrations above 50 mu M, bilirubin expos
ure resulted in a 71 +/- 8% (mean +/- SD) inhibition of kinase activit
y (p < 0.001, t test, n = 10). Bilirubin exposure did not results in k
inase inhibition if excessive bilirubin was removed by albumin binding
before stimulation of kinase activity (106.9 +/- 8.6% control activit
y, n = 5), However, removal of bilirubin by binding with albumin after
calcium addition did not restore kinase activity, (36.1 +/- 3.8% cont
rol activity, n = 5). Thus, once inhibition was observed, the activity
could not be restored by addition of albumin. The data suggest that b
ilirubin exposure resulted in a calcium-dependent inhibition of CaM ki
nase II activity that, once induced, was not reversible by removing bi
lirubin by the addition of albumin, Because inhibition of CaM kinase I
I activity has been correlated with delayed neuronal cell death in man
y neuropathologic conditions, bilirubin-induced inhibition of this enz
yme may be a cellular mechanism by which bilirubin exposure results in
delayed neuronal cell death in developing brain.