Decreased p38 MAPK activity in end-stage failing human myocardium: p38 MAPK alpha is the predominant isoform expressed in human heart.

Short duration exposure to cellular stresses have been shown to activate p38 mitogen-activated protein kinase (MAPK) in cultured rat ventricular cardiomyocytes and isolated perfused hearts; however, effects of chronic stress on p38 MAPK are not well understood. This study determined whether alterations in the p38 MAPK pathway occurred prior to end-stage human heart failure. The p38 MAPK alpha isoform was detectable in human cardiac tissue. However, carefully controlled analysis of protein and message in this study demonstrated an absence of the p38 MAPK beta -isoform. Low levels of message for the non-SB203580 sensitive p38 MAPK gamma and delta isoforms were also detected in both normal and failing human myocardium. Ischemic and idiopathic end-stage failing human hearts were compared to non-failing hearts for both p38 alpha MAPK protein level and total p38 MAPK activity. Western blotting techniques demonstrated no significant changes in total p38 alpha MAPK content. However, approximately 75% decreases in active/phosphorylated p38 MAPK (P<0.005) were observed in both ischemic and idiopathic failing hearts compared to non-failing hearts. In-gel kinase assays confirmed that activated p38 MAPK, detected by Western blotting, phosphorylated its potential downstream targets. When compared to non-failing hearts, approximately 46% decreases in p38 MAPK phosphorylation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2) were observed in ischemic and idiopathic failing hearts (P=0.03 and P=0.04 respectively). Active p38 MAPK was localized to sarcomeric structures in the cytosol of myocytes by confocal immunofluorescence microscopy. The correlation between decreased MAPKAPK-2 phosphorylation and loss of active p38 MAPK in failing human myocytes suggests that decreases in the activation of p38 MAPK alpha, the predominant cardiac isoform, occur prior to end-stage heart failure.

Wortmannin inhibits the growth of mammary tumors despite the existence of a novel wortmannin-insensitive phosphatidylinositol-3-kinase.

PURPOSE: Phosphatidylinositol (PtdIns) 3-kinase is an important mediator of many cellular functions. The study of PtdIns 3-kinase has been facilitated by the existence of the potent irreversible inhibitor of p110 PtdIns 3-kinase, wortmannin. The purpose of the study was to investigate the relationship between the cell growth inhibitory activity and antitumor activity of wortmannin and inhibition of PtdIns 3-kinase. METHODS: PtdIns 3-kinase activity was measured in cells and tumors and the effects of wortmannin investigated. RESULTS: Wortmannin inhibited the growth of murine C3H and human MCF-7 mammary tumors in vivo. However, the ability of wortmannin to inhibit C3H tumor growth was not related to inhibition of tumor PtdIns 3-kinase activity. The existence of wortmannin-insensitive PtdIns 3-kinase activity was demonstrated in C3H and MCF-7 cell culture lysates and solid tumors, and normal mouse tissue homogenates. In addition to being resistant to inhibition by wortmannin, MCF-7 cell lysate total PtdIns 3-kinase activity was also resistant to five additional known inhibitors of p110 PtdIns 3-kinase. Partial purification of wortmannin-insensitive PtdIns 3-kinase from MCF-7 cell lysate showed the activity to be independent of the PtdIns 3-kinase p85 regulatory subunit. CONCLUSION: The results of the current study demonstrate that wortmannin can inhibit the growth of murine and human mammary tumors despite the presence of novel wortmannin-insensitive PtdIns 3-kinases in these tissues suggesting that some other target is responsible for wortmannin's antitumor activity.

Acetylation and its role in the mutagenicity of the antihypertensive agent hydralazine.

1-Hydrazinophthalazine [hydralazine (HDZ)] is a hydrazine derivative that is a direct acting vasodilator effective in the treatment of essential hypertension. HDZ is biotransformed by the phase II conjugation enzyme N-acetyltransferase (NAT) forming acetyl HDZ, which spontaneously cyclized to the stable product 3-methyl-s-triazolo- [3,4-alpha]-phthalazine (MTP). Therapeutic use of HDZ has resulted in adverse side effects, specifically a drug-induced systemic lupus erythematosus. Slow acetylators are more likely than rapid acetylators to develop this toxicity. Bacteria expressing different levels of NAT were used to test the hypothesis that acetylation of HDZ decreases its mutagenic potential. The variation in NAT activities was confirmed by incubating bacterial cultures with HDZ, and the formation of MTP was monitored by HPLC. At 1.0 mg/ml HDZ, YG1029 (NAT overexpresser) produced 5.3 times the amount of MTP as TA100 (normal NAT expresser), and this production was linear for 20 hr. In the Salmonella mutagenesis assay, HDZ produced a dose- and strain-dependent increase in the number of revertants observed. Exposure to 4 mg HDZ/plate resulted in 1000 revertants in the overexpressing strain, YG1029, whereas both TA100 and TA100/1,8DNP6, which express normal levels and lack the NAT protein respectively, produced 1600 revertants. Colony hybridization analysis using probes for each of the six possible TA100 reverting mutations was performed to determine the nature of the mutations. The G:C to T:A transversion was the only mutation whose frequency was increased significantly by HDZ. Fifty-four percent of the induced vs. 25% of the spontaneous mutations were C to A transversions.(ABSTRACT TRUNCATED AT 250 WORDS)