111 - Effects of Bilirubin on the Distribution of Large Conductance Calcium and Voltage-activated K+ Channels (BK) in Lipid Rafts of the Nucleus Tractus Solitarius.

Apnea of prematurity (AOP) is a common cause of short- and long-term morbidity in preterm infants. There is a growing body of evidence supporting the association of apnea in neonates with hyperbilirubinemia. Our group has previously shown that bilirubin can cause damage to the cerebellum, an area of rapid growth in preterm infants, by localizing to dynamic membrane microdomains known as lipid rafts (LR) and disrupting signal transduction and protein trafficking in the Gunn rat model. The homozygous Gunn rat (jj) lacks the ability to conjugate bilirubin which results in the accumulation of unconjugated bilirubin bound to albumin. Administration of sulfadimethoxine (SDMX) results in acute elevation of free bilirubin, which can cross the blood brain barrier and perturb developing regions of the brain. Another area of rapid development in preterm infants is the nucleus tractus solitarius (NTS), which is implicated in the control of respiratory drive. The large conductance calcium and voltage-activated K⁺ channels (BK) are lipid raft associated proteins that are important in regulating the neuronal activity of the NTS.

Objective:

To study the effects of bilirubin on the lipid raft distribution of BK in the NTS in a rat model of preterm hyperbilirubinemia.

Design/Methods:

On postnatal day (P) 5, heterozygous (Nj) and jj Gunn rat pups were treated intraperitoneally with 200 mg/kg SDMX or an equivalent volume of saline (n=3 per group). P5 rat pups are equivalent to 25 – 29 week preterm infants. The NTS was dissected out of the brainstem on P6. The NTS tissue was then homogenized in low detergent lysis buffer and overlaid by a sucrose density gradient. The gradient was centrifuged at 180,000 g for 24 hours at 4^oC. Sequential fractions were drawn off the top of the gradient. Fractions containing LR were identified by western blot for cholera toxin subunit B and were combined into LR and non-LR (N) pools. Both pools were precipitated using chloroform/methanol, resuspended in Laemmli sample buffer, run on SDS-PAGE, and immunoblotted for BK.

Results:

The percent of BK found in LR of P6 jj Gunn rat pup NTS is significantly reduced after treatment with SDMX as compared to jj pups treated with saline (p=0.036, ANOVA Tukey post hoc). The percent of BK found in LR of P6 Nj Gunn rats was not affected by treatment with SDMX.

Conclusion(s):

The acute elevation of free bilirubin in P5 Gunn rats results in reduction of the percent of BK in lipid rafts. These results suggest one mechanism through which bilirubin may perturb neuronal activity of the NTS leading to AOP in preterm infants.