Cold-induced protein tyrosine phosphorylation in cerebral arteries of newborn lambs: a mechanism of cold-induced contraction • 1177

Hypothermia is widely utilized in clinical practice, i.e., cardiac surgery and organ preservation, and has been proposed for management of brain injury. Cerebrovascular dysfunction associated with hypothermic cardiopulmonary bypass suggests a potential negative impact of hypothermia. We have recently reported that a calcium-dependent cold-induced contraction of middle cerebral arteries(MCA) from newborn lambs possibly linked to protein tyrosine kinase(PTK)-/protein tyrosine phosphatase(PTP)-dependent signal transduction pathways. The present study evaluated changes in protein tyrosine phosphorylation in conjunction with contractile behavior of MCA preparations subjected to deep hypothermia (17°-12°C) in organ bath (Kreb's buffer, 21% O₂, 5% CO₂). MCA rings (n=10) mounted for isometric force recording showed significant increase in force generation from 0.78±0.01 grams at 37°C to maximum of 1.29±0.12 grams at 12°C. For biochemical analysis, control and cold-challenged MCA segments were rapidly frozen in liquid N₂ and analyzed using standard electrophoretic and immunoblotting techniques. Protein tyrosine phosphorylation was probed with a monoclonal anti-phosphotyrosine antibody conjugated with horse-radish peroxidase, detected by enhanced chemiluminescence, and densitometric analysis. Cold-challenged preparations expressed 2- to 5-fold increase in protein tyrosine phosphorylation(reproduced in three animals). The most prominent cold-sensitive phosphotyrosyl-proteins detected were in the 90 kDa, 70 kDa, and 60 kDa range. Both cold-induced tyrosine phosphorylation and contraction were reduced by genistein (20-100 μM), a nonselective PTK inhibitor, and blocked by damnacanthal (1 μM), a selective inhibitor of the non-receptor src-family of PTKs (56-60 kDa proteins). The data demonstrate a cold-induced contraction of MCA's associated with cold-induced protein tyrosine phosphorylation. PTK regulation of calcium homeostasis has been reported. Our findings provide evidence for cold-sensitive PTK/PTP-signal transduction machinery, possibly linked to calcium homeostasis. This pathway may be a key factor in the temperature-dependent alteration of contractile behavior and previously reported cerebrovascular dysfunction associated with hypothermia.