Influence of glutathione levels and activity of glutathione-related enzymes in the brains of tumor-bearing mice.

Oxidative stress takes place due to an imbalance between the production of reactive oxygen species (ROS) and the protection provided by cellular antioxidants. High levels of ROS are caused by tumor cells during tumor progression and may affect the functions of other important organs. The present study sought to investigate whether non-primary brain tumors affect reduced glutathione (GSH) levels and the activity of related enzymes in the brain. GSH contents, the activity of glutathione peroxidase (GPx), glutathione-s-transferase (GST), glutathione reductase (GR) as well as glutamate cysteine ligase (GCL) were determined in the brains of normal and tumor-bearing mice treated with the chemotherapy drug 5-Fluorouracil (5-Fu) or not. The results in S180 and H22 tumor-bearing mice showed that GSH levels and the activity of GPx, GST, and GCL all decreased while GR activity markedly increased in the brains of tumor-bearing mice compared to those of normal mice. Further investigation found that 5-Fu, a typical chemotherapy drug, significantly inhibited tumor growth but did not improve the loss of redox homeostasis in the brain caused by non-primary brain tumors. Overall, these results suggest that non-primary brain tumors can induce an ROS burden in the brain that cannot be reversed by the chemotherapy drug 5-Fu.

The amino terminus with a conserved glutamic acid of G protein-coupled receptor kinases is indispensable for their ability to phosphorylate photoactivated rhodopsin.

To investigate functions of the consensus amino terminus of G protein-coupled receptor kinases (GRKs), two amino terminus-truncated mutants (delta30 or delta15) and two single-amino-acid mutants of conserved acidic residues (D2A or E7A) of human GRK1 were constructed and expressed in human embryonic kidney 293 cells. It was shown that truncated mutations and one single-point mutation (E7A) greatly decreased GRK1's activity to phosphorylate photoactivated rhodopsin (Rho*), whereas the abilities of these mutants to phosphorylate a synthetic peptide substrate and to translocate from cytosol to rod outer segments on light activation were unaffected. Further experiments demonstrated that the same truncated mutations (delta30 or delta15) of GRK2, representative of another GRK subfamily, also abolished the kinase's activity toward Rho*. The similar single-point mutation (E5A) of GRK2 heavily impaired its phosphorylation of Rho* but did not alter its ability to phosphorylate the peptide, and the G329-rhodopsin-augmented peptide phosphorylation by GRK2 (E5A) remained unchanged. Our data, taken together, suggest that the amino terminus as well as a conserved glutamic acid in the region of GRKs appears essential for their ability to functionally interact with G protein-coupled receptors.

Selective interference of beta-arrestin 1 with kappa and delta but not mu opioid receptor/G protein coupling.

The role of beta-arrestin 1 (beta-arr1) in regulation of responsiveness of kappa, delta, and mu opioid receptors has been investigated in human embryonic kidney 293 cells cotransfected with opioid receptor and beta-arr1. Expression of human beta-arr1 attenuated kappa and delta opioid receptor subtype-mediated inhibition of cAMP production and resulted in a 100-fold increase of EC50 values for kappa-agonist U69593 and delta-agonist [D-Pen2, D-Pen5]enkephalin and 30-40% reduction of their maximal responses. In contrast, coexpression of beta-arr1 with mu opioid receptor did not affect the concentration-effect relationship of mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin. In parallel, kappa and delta receptor-mediated G protein activation was also remarkably attenuated by overexpression of beta-arr1, while the mu-agonist-stimulated response remained intact. These results indicate that beta-arr1 interferes receptor/G protein coupling and differentially regulates the responsiveness of opioid receptors. Truncation of kappa and delta opioid receptors at carboxyl termini abolished inhibition of beta-arr1 on the responsiveness of both receptors. Furthermore, mu opioid receptor became sensitive to beta-arr1 regulation following replacement of its carboxyl terminus with the corresponding portion of the delta receptor. Removal of potential phosphorylation sites on the carboxyl terminus of kappa opioid receptor led to reduced effect of beta-arr1 on the receptor-mediated response. These results suggest that receptor carboxyl terminus and its phosphorylation play an important role in the interaction of beta-arr1 and opioid receptors.

Carboxyl terminal of rhodopsin kinase is required for the phosphorylation of photo-activated rhodopsin.

Human rhodopsin kinase (RK) and a carboxyl terminus-truncated mutant RK lacking the last 59 amino acids (RKC) were expressed in human embryonic kidney 293 cells to investigate the role of the carboxyl terminus of RK in recognition and phosphorylation of rhodopsin. RKC, like the wild-type RK, was detected in both plasma membranes and cytosolic fractions. The C-terminal truncated rhodopsin kinase was unable to phosphorylate photo-activated rhodopsin, but possesses kinase activity similar to the wild-type RK in phosphorylation of small peptide substrate. It suggests that the truncation did not disturb the gross structures of RK catalytic domain. Our results also show that RKC failed to translocate to photo-activated rod out segments. Taken together, our study demonstrate the carboxyl terminus of RK is required for phosphorylation of photo-activated rhodopsin and strongly indicate that carboxyl-terminus of RK may be involved in interaction with photo-activated rhodopsin.