phosphothreonine and Hypoxia

The roles of Rho kinase (ROCK) and cGMP-dependent protein kinase (PKG) in cGMP-mediated relaxation of fetal pulmonary veins exposed to chronic hypoxia (CH) were investigated. Fourth generation pulmonary veins were dissected from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days (CH) and from control ewes. After constriction with endothelin-1, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) caused a similar relaxation of both control and CH vessels. Rp-8-Br-PET-cGMPS (a PKG inhibitor) inhibited whereas Y-27632 (a ROCK inhibitor) augmented relaxation of control veins to 8-Br-cGMP. These effects were significantly diminished in CH veins. PKG protein expression and activity were greater whereas ROCK protein expression and activity were less in CH vessels compared with controls. Phosphorylation of threonine 696 (ROCK substrate) and serine 695 (PKG substrate) of the regulatory myosin phosphatase targeting subunit MYPT1 of myosin light chain (MLC) phosphatase was stimulated to a lesser extent in CH than in control veins by endothelin-1 (ROCK stimulant) and 8-Br-cGMP (PKG stimulant), respectively. The phosphorylation and dephosphorylation of MLC caused by endothelin-1 and 8-Br-cGMP, respectively, were less in CH veins than in controls. These results suggest that CH in utero upregulates PKG activity but attenuates PKG action in fetal pulmonary veins. These effects are offset by the diminished ROCK action on MYPT1 and MLC and thus lead to an unaltered response to cGMP.

Topics: Altitude; Animals; Chronic Disease; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Female; Fetus; Hypoxia; In Vitro Techniques; Models, Biological; Myosin Light Chains; Myosin-Light-Chain Kinase; Phosphorylation; Phosphoserine; Phosphothreonine; Pulmonary Veins; rho-Associated Kinases; Sheep; Vasodilation

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase are protein-serine/threonine kinases which are important regulators of diverse cellular processes including metabolism, proliferation and differentiation. This study shows that both hypoxia and X irradiation of serum-deprived Chinese hamster V79 cells cause the induction and phosphorylation of the PKC-alpha isoform. The increased induction and phosphorylation of PKC occur mainly in the nuclear fraction. Unlike the PKC activator TPA, neither hypoxic nor radiation stress causes translocation of PKC-alpha from the cytosol to the membrane. The induction of PKC-alpha by hypoxia is accompanied by an increased expression of MAP kinase but, in contrast, this does not occur when PKC-alpha is induced by radiation. Radiation, like TPA, causes a complete redistribution of MAP kinase from the cytosol to the nucleus.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Compartmentation; Cell Line; Cricetinae; Cricetulus; Enzyme Induction; Hypoxia; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Kinase C; X-Rays