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.
Funded by a grant from the American Heart Association
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Wagerle, L., Dahdah, N. & Russo, P. Cold-induced protein tyrosine phosphorylation in cerebral arteries of newborn lambs: a mechanism of cold-induced contraction • 1177. Pediatr Res 43, 202 (1998). https://doi.org/10.1203/00006450-199804001-01198