Bilirubin Induces a Calcium-Dependent Inhibition of Multifunctional Ca2+/Calmodulin-Dependent Kinase II Activity in Vitro

Abstract

Excessive bilirubin levels in newborn infants result in longterm neurologic deficits that remain after bilirubin levels return to normal. Much of the observed neurologic deficits can be attributed to bilirubin-induced, delayed neuronal cell death. Inhibition of calcium/calmodulin-dependent kinase I1 (CaM kinase 11) activity that precedes cell death is observed in conditions such as seizure activity, stroke, and glutamate 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 I1 activity, in vitro. P-81 filtration assay of basal and calcium-stimulated kinase activity was performed under standard kinase assay conditions. Bilirubin and/or albumin was added to the reaction vessels to determine the effect of these agents on kinase activity. Bilirubin exposure resulted in a concentration-dependent inhibition of CaM kinase I1 activity (IC,, = 16.78 pM). At concentrations above 50 pM, bilirubin exposure resulted in a 71 ? 8% (mean ? SD) inhibition of kinase activity (p < 0.001, t test, n = 10).Bilirubin exposure did not result in kinase inhibition if excessive bilirubin was removed by albumin binding before stimulation of kinase activity (106.9 ? 9.6% control activity, n = 5). However, removal of bilirubin by binding with albumin after calcium addition did not restore kinase activity. (36.1 5 3.8% control activity, n = 5). Thus, once inhibition was observed, the activity could not be restored by addition of albumin. The data suggest that bilirubin exposure resulted in a calcium-dependent inhibition of CaM kinase I1 activity that, once induced, was not reversible by removing bilirubin by the addition of albumin. Because inhibition of CaM kinase I1 activity has been correlated with delayed neuronal cell death in many neuropathologic conditions, bilirubin-induced inhibition of this enzyme may be a cellular mechanism by which bilirubin exposure results in delayed neuronal cell death in developing brain. (Pediatr Res 38: 949-954, 1995) Abbreviation CaM kinase 11, calcium/calmodulin-dependent kinase I1

The Effects of Bilirubin on Evoked Potentials and Long-Term Potentiation in Rat Hippocampus In Vivo

“…In the meantime, the protein-kinase system is essential for maintaining the LTP by auto-phosphorylation. Recent in vitro studies have showed that bilirubin has extensive inhibitory effects on many protein kinases (2,22). This might explain the finding of an inhibitory effect of bilirubin on the inducing process of LTP in our in vivo study.…”

Bilirubin and the Auditory System

“…The activity of the enzyme calcium/ calmodulin kinase II (CaM-Kinase II) is inhibited in vitro by physiologic doses of calcium. 13 Using cultured hippocampal pyramidal neurons, we found increased intracellular calcium occurs immediately after micropipeting a solution containing 32 mol/l bilirubin plus 32 mol/l human serum albumin. The increase in intracellular calcium peaks within a minute and then decreases to a steady state slightly above baseline approximately 45 minutes after exposure.…”

Minocycline blocks bilirubin neurotoxicity and prevents hyperbilirubinemia‐induced cerebellar hypoplasia in the Gunn rat

“…Mitochondrial membranes obtained from neurons oxidize bilirubin at a significantly lower rate than membranes prepared from a mixed glial ⁄ neuronal source, suggesting that neurons may be less able to detoxify bilirubin which might contribute to the apparent higher sensitivity of neurons to bilirubin toxicity than glia (Hansen & Allen, 1997). Additionally, bilirubin has been reported to inhibit calcium-mediated calmodulin kinase activity (Churn et al, 1995). None of these findings, however, explain the selective vulnerability of neurons, especially cerebellar neurons, to bilirubin toxicity.…”