Abstract
The present study aims to investigate the dependence of CaM kinase IV cascade activation during hypoxia and tests the hypothesis that hypoxia-induced tyrosine phosphorylation of CaM and CaM kinase IV, activation of CaM kinase IV and phosphorylation of CREB protein during hypoxia increases as a function of increase in cerebral tissue hypoxia as measured by decrease in tissue ATP and phosphocreatine (PCr). 3-5 days old newborn piglets were divided into normoxic (Nx, FiO₂ of 0.21 for 1h) and hypoxic (Hx, FiO₂ of 0.07 for 1h) groups. Cerebral tissue hypoxia was documented by determining the levels of high energy phosphates ATP and phosphocreatine (PCr). Cerebral cortical neuronal nuclei were isolated and purified, and tyrosine phosphorylation of calmodulin (Tyr⁹⁹), the activator of CaM kinase IV, and CaM kinase IV determined by Western blot using anti-phospho-(pTyr⁹⁹)-calmodulin, anti-pTyrosine and anti-CaM kinase IV antibodies. The activity of CaM kinase IV and its consequence the phosphorylation of CREB protein at Ser¹³³ were determined. The levels of ATP (μmole/g brain) ranged from 3.48 to 5.28 in Nx, and 0.41 to 2.26 in Hx. The levels of PCr (μmole/g brain) ranged from 2.46 to 3.91 in Nx and 0.72 to 1.20 in Hx. The pTyr⁹⁹ calmodulin (OD x mm²) ranged from 20.35 to 54.47.60 in Nx, and 84.52 to 181.42 in Hx (r²=0.5309 vs ATP and r²=0.6899 vs PCr). Expression of tyrosine phosphorylated CaM kinase IV ranged from 32.86 to 82.46 in Nx and 96.70 to 131.62 in Hx (r²=0.5132 vs ATP and r²=0.4335 vs PCr). The activity of CaM kinase IV (pmole/mg protein/min) ranged from 1263 to 3448 in Nx and 3767 to 6633 in Hx (r²=0.7113 vs ATP and r²=0.6182 vs PCr). The expression of p-CREB at Ser¹³³ ranged from 44.26 to 70.28 in Nx and 82.70 to 182.86 in Hx (r²=0.6621 vs ATP and r²=0.5485 vs PCr). The data show that hypoxia results in increased tyrosine phosphorylation of calmodulin (Tyr⁹⁹), increased tyrosine phosphorylation of CaM kinase IV, increased activity of CaM kinase IV and increased phosphorylation of CREB at Ser¹³³ as an inverse function of cerebral concentration of high energy phosphates, ATP and PCr. We conclude that the hypoxia-induced increased activation of CaM kinase IV cascade increases with the increase in the degree of cerebral tissue hypoxia as measured by cerebral tissue high energy phosphates in a curvilinear manner. The tyrosine kinases (Src kinase and EGFR kinase) mediated activation of CaM kinase IV cascade potentially results in increased CREB phosphorylation that triggers transcription of proapoptotic proteins during hypoxia.