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1 Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA
2 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA
* To whom correspondence should be addressed. E-mail: kgk{at}po.cwru.edu.
The regulation of tyrosine hydroxylase (TH) by intermittent hypoxia (IH) was investigated in rat pheochromocytoma 12 (PC12) cells. Cells were exposed to alternating cycles of hypoxia (1% O2; 15 sec) and normoxia (21% O2; 3 min) for up to 60 cycles while controls were exposed to normoxia for a similar duration. IH exposure increased both dopamine content and TH activity by ~ 42 and ~56 %, respectively. Immunoblot analysis revealed that comparable levels of TH protein were expressed in normoxic and IH cells. Removal of TH bound catecholamines and in vitro phosphorylation of TH in cell-free extracts by the catalytic subunit of protein kinase A (PKA) increased TH activity in normoxic but not in IH cells suggesting possible induction of TH phosphorylation and removal of endogenous inhibition of TH by IH. To assess the role of serine phosphorylation in IH-induced TH activation, TH immunoprecipitates and extracts derived from normoxic and IH cells were probed with anti-phosphoserine and anti-phospho TH (ser-40) antibody, respectively. Compared to normoxic cells, both total serine and serine-40 specific phosphorylation of TH were increased in IH cells. IH-induced activation of TH and the increase in total serine and serine-40 specific phosphorylation of TH were inhibited by Ca2+-calmodulin dependent protein kinase (CaMK) and PKA specific inhibitors but not by inhibitor of extracellular signal regulated protein kinase pathway. Taken together, these observations suggest that intermittent hypoxia activates TH in PC12 cells via phosphorylation of serine residues including serine-40, in part, by CaMK and PKA. Our results also suggest that intermittent hypoxia-induced phosphorylation of TH facilitates the removal of endogenous inhibition of TH leading to increased synthesis of dopamine.
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