Effect of cerebral hypoxia on NMDA receptor binding characteristics after treatment with 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) in newborn piglets.
Previous studies have shown that hypoxia modifies the NMDA receptor/ion channel complex in cortical brain cell membranes of newborn piglets. The present study tests the hypothesis that blockade of the glutamate recognition site of the NMDA receptor with the competitive antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) prevents modification of the receptor during hypoxia. Twenty seven anesthetized, ventilated newborn piglets were randomized into four groups: 7 normoxic (Nx), 6 CPP-treated normoxic (CPP-Nx), 8 hypoxic (Hx) and 6 CPP-treated hypoxic (CPP-Hx). Treatment groups received CPP 2 mg/kg i.v. The CPP-Hx group received CPP 30 min prior to hypoxia, which was induced by lowering the FiO2 to 5-7% for 1 h. Physiologic data showed no change in heart rate, blood pressure, arterial blood gas values, glucose or lactate following CPP administration. During hypoxia there was a significant decrease in PaO2, pH and an increase in lactate compared to baseline values. The CPP-Hx group had significantly higher lactate levels than the Hx group during hypoxia. P2 membrane fractions were prepared and thoroughly washed. Characteristics of the NMDA receptor ion channel were determined by [3H]MK-801 binding assays and characteristics of the glutamate recognition site by specific NMDA-displaceable [3H]glutamate binding assays. Brain tissue ATP and PCr levels confirmed tissue hypoxia, and were not preserved by CPP administration. [3H]MK-801 binding assays revealed that CPP treatment attenuated the hypoxia-induced decrease in the number of receptors (Bmax) and receptor binding affinity (Kd) during hypoxia. CPP treatment also decreased receptor affinity (increased Kd) for [3H]MK-801 binding during normoxia and hypoxia. Assays of [3H]glutamate binding revealed that hypoxia decreased both the Bmax and the Kd of the NMDA receptor for [3H]glutamate and both were preserved by CPP treatment prior to hypoxia. CPP had no effect on [3H]glutamate Bmax or Kd during normoxia. We conclude that hypoxia decreases the Bmax and Kd of the NMDA receptor glutamate recognition site for [3H]glutamate and the ion channel site for [3H]MK-801 in newborn piglets. These changes are prevented by CPP administration prior to hypoxia. The different effects of CPP binding during normoxia and hypoxia suggest a use-dependent mechanism for CPP binding during hypoxia, possibly through an hypoxia-induced alteration of the high-affinity binding site for CPP. During both normoxia and hypoxia CPP binding appeared to induce a conformational change in the receptor causing a decrease in binding affinity for [3H]MK-801. CPP administration did not preserve brain tissue ATP or PCr levels during hypoxia and may alter cellular metabolism in addition to its action at the NMDA receptor. However, even with depletion of the energy precursors ATP and PCr, and with higher lactate levels in the CPP-Hx group, CPP was able to maintain NMDA receptor binding characteristics during hypoxia and may decrease excitotoxic cellular damage from hypoxia.
Fritz
K I
KI
Department of Pediatrics, Thomas Jefferson University Hospital, Philadelphia, PA 19107, USA.
Groenendaal
F
F
McGowan
J E
JE
Mishra
O P
OP
Delivoria-Papadopoulos
M
M
D000818:Animals; D000831:Animals, Newborn; D004353:Drug Evaluation, Preclinical; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D002534:Hypoxia, Brain / Q000378:metabolism*; D016014:Linear Models; D010879:Piperazines / Q000494:pharmacology*; D011897:Random Allocation; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012016:Reference Values; D013552:Swine
D018691:Excitatory Amino Acid Antagonists; D010879:Piperazines; D016194:Receptors, N-Methyl-D-Aspartate; C050592:3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid