Calphostin A

1. In inflammatory kidney diseases procoagulatory activity (PCA) becomes evident. Glomerular fibrin deposits and capillary microthrombi are histopathological hallmarks in most forms of glomerulonephritis. 2. Therefore in this study the expression of tissue factor (TF) as the main inducer of thrombogenesis was examined in cultured human mesangial cells (MC) in response to proinflammatory stimuli such as interleukin-1 (IL-1 beta), tumour necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS). Also main signalling pathways were investigated. 3. IL-1 beta, TNF-alpha and LPS induced TF in MC in a time and dose dependent manner on mRNA and protein levels. Highest activity was found after 12 h of stimulation. Induction of TF was completely blockable by BAPTA-AM, a chelator of intracellular [Ca(2+)](i) as well as calphostin, a protein kinase C (PKC) inhibitor. Activation of the protein kinase A (PKA) pathway had no influence on basal TF expression, but down-regulated cytokine-induced TF. The PKA blocker, KT5720, increased TF formation significantly. Since TF exerts its activity primarily on the surface of cells and after release of encrypted receptors we further tested TF activity in MC supernatants. IL-1 beta did not significantly increase TF activity in supernatants of intact cells. However, when MC were rendered apoptotic by oxidative metabolites, IL-1 beta treated MC released highly stimulated TF activity into the supernatants, suggesting that a paracrine activation of the coagulatory cascade can take place under such conditions. 4. Inflammatory mediators up-regulate TF expression in MC by a PKC dependent pathway whereas PKA can serve as a negative feed-back link. Apoptosis of inflammatory MC may trigger to spread PCA.

Dietary supplementation with fish oil that contains omega-3 polyunsaturated fatty acids has been shown to enhance bone density as well as duodenal calcium uptake in rats. The latter process is supported by membrane ATPases. The present in vitro study was undertaken to test the effect of omega-3 fatty acids on ATPase activity in isolated basolateral membranes from rat duodenal enterocytes. Ca-ATPase in calmodulin-stripped membranes was activated in a biphasic manner by docosahexanoic acid (DHA) (10-30 microg/ml) but not by eicosapentanoic acid (EPA). This effect was blocked partially by 0.5 microM calphostin (a protein kinase C blocker). DHA inhibited Na,K-ATPase (-49% of basal activity, [DHA]=30 microg/ml, P <0.01). This effect could be reversed partially by 50 microM genistein, a tyrosine kinase blocker. EPA also inhibited Na,K-ATPase: (-47% of basal activity, [EPA]=30 microg/ml, P <0.01), this effect was partially reversed by 100 microM indomethacin, a cyclo-oxygenase blocker. Omega-3 fatty acids are thus involved in multiple signalling effects that effect ATPases in BLM.

The expression of genes that regulate cell growth, such as ornithine decarboxylase (ODC), can be modulated by oxidant tumor promoters. Treatment of murine papilloma PE cells with H2O2 led to a transient induction of ODC enzyme activity, which could be blocked by calphostin, a nonspecific inhibitor of protein kinase C (PKC). Peak activity (11-fold) occurred 5-6 h after treatment, followed by a rapid decline. The increase in ODC activity was associated with an elevation of both ODC mRNA (3-fold) and protein (7-fold). Direct involvement of PKC in the regulation of ODC by oxidants was determined by stable transfection of PE cells with a dominant-negative PKC-delta mutant. PKC-delta activity was completely inhibited in response to H2O2 in cells overexpressing mutant PKC-delta compared with cells transfected with a blank plasmid. Induction of ODC mRNA, protein, and activity was also completely inhibited in cells expressing the PKC-delta mutant after H2O2 treatment. Activation of an ODC promoter-luciferase reporter construct by H2O2 was attenuated in mutant cells compared with control cells, further confirming that ODC is regulated transcriptionally by PKC-delta. However, fold-increases in ODC mRNA and protein were much less than the increase in activity, suggesting that ODC may also undergo posttranscriptional regulation in the presence of oxidants. Taken together, these studies provide new insight into the regulation of ODC by oxidants and suggest that PKC-delta may play a critical role in this regulation.