Deficient nifedipine oxidation: a rare inherited trait associated with cystic fibrosis kindreds.

Previous studies have indicated that there is weak genetic linkage between the defective gene in cystic fibrosis (CFTR) and the gene encoding the nifedipine metabolizing enzyme P4503A4 which are both located on chromosome 7. To examine further this possible association, nifedipine metabolism was investigated in a group of 59 volunteers, and 17 adult cystic fibrosis patients and 37 of their relatives. In agreement with the majority of previous studies, the volunteer group showed a unimodal distribution of recoveries for the major metabolite M-II ranging from 33 to 78% excretion in 8 h. In the case of both the cystic fibrosis patients and their parents, the distribution of recoveries was shifted to the left with five out of 20 parents and three out of 11 unrelated cystic fibrosis patients showing recoveries below the range observed in the volunteer group. This poor metabolism appeared to be both reproducible and heritable and did not appear to be a consequence of mutations in the CFTR gene.

Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase.

Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2 in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities.

In vivo regulation of protein-serine kinases by insulin in skeletal muscle of fructose-hypertensive rats.

The effects of tail-vein insulin injection (2 U/kg) on the regulation of protein-serine kinases in hindlimb skeletal muscle were investigated in hyperinsulinemic hypertensive fructose-fed (FF) animals that had been fasted overnight. Basal protein kinase B (PKB) activity was elevated about twofold in FF rats and was not further stimulated by insulin. Phosphatidylinositol 3-kinase (PI3K), which lies upstream of PKB, was increased approximately 3.5-fold within 2-5 min by insulin in control rats. Basal and insulin-activated PI3K activities were further enhanced up to 2-fold and 1.3-fold, respectively, in FF rats. The 70-kDa S6 kinase (S6K) was stimulated about twofold by insulin in control rats. Both basal and insulin-stimulated S6K activity was further enhanced up to 1.5-fold and 3.5-fold, respectively, in FF rats. In control rats, insulin caused a 40-50% reduction of the phosphotransferase activity of the beta-isoform of glycogen synthase kinase 3 (GSK-3beta), which is a PKB target in vitro. Basal GSK-3beta activity was decreased by approximately 40% in FF rats and remained unchanged after insulin treatment. In summary, 1) the PI3K --> PKB --> S6K pathway was upregulated under basal conditions, and 2) insulin stimulation of PI3K and S6K activities was enhanced, but both PKB and GSK-3 were refractory to the effects of insulin in FF rats.

PD98059 attenuates hydrogen peroxide-induced cell death through inhibition of Jun N-Terminal Kinase in HT29 cells.

We have investigated the effects of hydrogen peroxide (H(2)O(2)), a potent naturally occurring oxidant on cell signaling and viability in the pluripotent HT29 intestinal cell line. There was a dose-dependent reduction in cell viability upon exposure to H(2)O(2) as measured by the XTT assay. Features of apoptosis were indicated by the findings of PARP and caspase 3 cleavage, as well as changes in cell morphology using phase contrast and nuclear fragmentation using fluorescence microscopy. There was a dose-dependent increase in the activation of p45-JNK, p42/p44-ERK, and p38-HOG. Surprisingly, oxidant-induced cell injury could be attenuated by preincubation with PD98059 to 50% of untreated control cells (P = 0.002). This and UO126, another MEK inhibitor were ably to reproducibly inhibit p45-JNK activation induced by hydrogen peroxide. Transfection with kinase-inactive constructs of JNK and ERK revealed that the improvement in cell viability was due to inhibition of JNK and not ERK. Transient transfections with AP-1 and NF-kappaB luciferase reporter constructs did not reveal any transcriptional activation due to hydrogen peroxide exposure however, in both cases the basal levels of transcriptional activity were suppressed in the presence of PD98059. It is concluded that JNK mediates H(2)O(2)-induced cellular injury in the HT29 cell line, and additionally, we report for the first time that JNK activation can be inhibited by both PD98059 and UO126 at conventional doses used to inhibit MEK.

Oxidized low density lipoprotein inhibits macrophage apoptosis through activation of the PI 3-kinase/PKB pathway.

Oxidized LDL (oxLDL) is known to induce endothelial adhesion molecule and monocyte chemoattractant protein 1 expression and this is thought to be involved in monocyte recruitment into atherosclerotic lesions. oxLDL has also been found to induce macrophage proliferation. The purpose of the present study was to determine whether oxLDL might also have the ability to increase macrophage populations by inhibiting apoptosis. We found that oxLDL caused a dose-dependent inhibition of the apoptosis that occurs in cultured bone marrow-derived macrophages after macrophage colony-stimulating factor (M-CSF) withdrawal without inducing proliferation. Incubation of macrophages with either native LDL or acetylated LDL had no effect on apoptosis. The prosurvival effect of oxLDL was not inhibited by neutralizing antibodies to granulocyte-macrophage colony-stimulating factor, was maintained in mice homozygous for a mutation in the M-CSF gene, and was not due to other secreted cytokines or growth factors. oxLDL caused activation of the mitogen-activated protein kinases ERK1/2 (extracellular signal-regulated kinases 1 and 2) as well as protein kinase B (PKB), a target of phosphatidylinositol 3-kinase (PI 3-kinase). Furthermore, there was phosphorylation of two important prosurvival PKB targets, I-kappaBalpha(Ser-32) and Bad(Ser-136). The MEK inhibitors PD 98059 and U0126 blocked ERK1/2 activation but did not diminish survival. Conversely, the PI 3-kinase inhibitors LY 294002 and wortmannin blocked PKB activation, and the ability of oxidized LDL to promote macrophage survival. Taken together, these results indicate that oxLDL can directly activate a PI 3-kinase/PKB-dependent pathway that permits macrophage survival in the absence of growth factors.