Mechanism of Protein Tyrosine Phosphorylation During

Hypoxia in the Cerebral Cortex of Newborn Piglets

Jeffrey M. Vinocur*

St. Christopher’s Hospital for Children

Research Day, June 8, 2001

* coauthored with Anita C. Truttmann, Syed H. Haider, Om P. Mishra, and Maria Delivoria-Papadopoulos

Background

• Hypoxic brain injury

• Apoptosis

• Phosphorylation

Hypothesis

Protein tyrosine phosphatase activity is decreased during hypoxia in

cortical neuronal cell membranes of newborn piglets.

Experimental ProtocolVentilated Newborn Piglets

NormoxiaFiO2: 0.21 for 1 hour

Acute HypoxiaFiO2: 0.05 - 0.07 for 1 hour

Membrane Preparation Biochemical Analysis

Cerebral Cortices Harvested

Anesthesia: Halothane 4%, Nitrous oxide, Tubocurarine (0.3 mg/kg), Fentanyl (50 ug/kg), Lidocaine 1%

Methods (1):Preparation of P2 Membranes

• Brain tissue was homogenized in a sucrose buffer.

• Homogenate was centrifuged at 1,000 g for 10 minutes, and subsequent supernatant at 40,000 g for 1 hour.

• Pellet was resuspended in HEPES buffer and assayed for protein content (Lowry et al., 1951).

Methods (2):ATP and PCr Determination

• Hypoxia can be documented by measuring a decrease in high-energy phosphates.

• Cerebral cortical tissue concentrations of ATP and PCr (phosphocreatine) were determined biochemically using the method of Lamprecht et al., 1974.

Methods (3):Protein Tyrosine Phosphatase Assay

p-nitrophenyl phosphate

(pNPP)

colorless yellowmax absorbance at 405 nm

ε = 20,000 cm-1 M-1

p-nitrophenol

(pNP)

phosphatase

dephosphorylation

Methods (3):Protein Tyrosine Phosphatase Assay

Total Inhibited

Phosphatase activity

Using bpV(phen), a specific inhibitor of protein tyrosine phosphatases, we can find PTP activity as the difference between total and inhibited activities.

Data:Documentation of Hypoxia

Study GroupATP

(umoles/g brain)

Phosphocreatine

(umoles/g brain)

Normoxia 4.57 ± 0.45 3.77 ± 0.36

Hypoxia 1.29 ± 0.23* 0.77 ± 0.13*

Decrease from Normoxia

71.9 % 79.6 %

Mean ± SD* p < 0.001 vs. normoxia

Data:Protein Tyrosine Phosphatase Activity

0

100

200

300

400

500

600

700

Normoxia HypoxiaPro

tein

Tyr

osin

e P

hosp

hata

se A

ctiv

ity

(nm

oles

pN

P/m

g pr

otei

n/hr

)

*

Mean ± SD* p < 0.001 vs. normoxia

525.20± 64.24

138.75± 35.79

Summary

Hypoxia results in significant decrease in protein tyrosine

phosphatase activity in cortical cell membranes of newborn piglets.

Speculation

• The decreased protein tyrosine phosphatase activity may be due to free radicals.

• Bcl-2 phosphorylation may prevent deactivation of Bax, leading to caspase-mediated apoptosis.