ABSTRACT
In this paper, we investigate the potential of a high-performance liquid chromatography technique to determine pKa values of drug candidates that show poor solubility in water. The determination of pKa values by this method is in principle not new, but it exhibits simplicity, requires lower quantities of drugs and solvents, and minimal analysis time. The method is an alternative to existing methodology, in which this determination is not readily feasible.
ABSTRACT
BACKGROUND: The cyclic adenosine 3',5'-monophosphate (cAMP) pathway plays a central role in the regulation of cell proliferation, differentiation and apoptosis. Cyclic AMP has been identified as a bifunctional regulator of apoptosis. The inhibitor of apoptosis proteins (IAP) regulates apoptosis by directly inhibiting distinct caspases. METHODS: Expression levels of rat IAP (RIAP)-1 were investigated by RNase protection assay in rat mesangial cells after stimulation with diverse agents that modulate cellular levels of cAMP. RESULTS: Rat mesangial cells up-regulated RIAP1 mRNA levels after cAMP stimulation. Membrane-permeable cAMP analogs, as well as cAMP production in response to the beta-adrenergic receptor agonist salbutamol caused a large increase in RIAP1 mRNA level, which could be inhibited by the protein kinase A inhibitors H89 and Rp-cAMPS, or by the nuclear factor-kappaB (NF-kappaB) inhibitor BAY117085. Inhibition of phosphodiesterase type IV by denbufyllin or rolipram potentiated the cAMP-mediated increase in RIAP1 mRNA. In contrast, the cyclic guanosine 3',5'-monophosphate (cGMP) analog Bt2cGMP did not affect the RIAP1 mRNA level. CONCLUSIONS: These data establish, to our knowledge for the first time, that RIAP1 mRNA levels are regulated by the cAMP-signaling pathway and suggest potential new avenues of therapy to modulate apoptosis.
Subject(s)
Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Glomerular Mesangium/metabolism , RNA, Messenger/metabolism , Viral Proteins/genetics , Animals , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic GMP/pharmacology , Enzyme Inhibitors/pharmacology , Glomerular Mesangium/cytology , Inhibitor of Apoptosis Proteins , RNA, Messenger/antagonists & inhibitors , Rats , Thionucleotides/pharmacology , Viral Proteins/metabolismABSTRACT
Mesangial cells and RAW 264.7 macrophages respond to different nitric oxide (NO) donors within 16 to 24 h or 6 to 8 h, respectively, with apoptotic cell death. RAW 264.7 macrophages also die in response to endogenous NO production. In contrast, endogenous NO production fails to significantly induce cell death in mesangial cells. It was hypothesized that differences in the expression of antiapoptotic proteins, in particular the inhibitor of apoptosis (IAP) protein family, might be responsible for this cell type-specific behavior. Therefore, IAP expression was investigated in relation to apoptosis induction in response to NO and cytokines in both cell types. In mesangial cells, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha induced cellular inhibitor of apoptosis 1 (cIAP1) mRNA expression within 3 h. In contrast, X chromosome-linked inhibitor of apoptosis (XIAP) mRNA levels remained unaffected by cytokines. Although coincubation of cells with IL-1beta and tumor necrosis factor-alpha or IL-1beta and basic fibroblast growth factor resulted in synergistic induction of inducible NO synthase, comparable potentiating effects on cIAP1 induction were absent. Exogenously released NO from NO donors promoted cIAP1 mRNA upregulation in mesangial cells, whereas XIAP mRNA was downregulated. However, the changes observed on the mRNA level were not adequately translated to the protein level, and corresponding values for cIAP1 and XIAP were only slightly affected. In contrast, in lipopolysaccharide/interferon-gamma-stimulated RAW 264.7 macrophages, massive NO-dependent downregulation of cIAP1 and XIAP protein levels, which correlated temporally with the induction of apoptosis, was observed. This effect was at least partially reversed by N(G)-monomethyl-L-arginine, an inhibitor of NO synthase activity. In summary, a direct correlation between the downregulation of IAP protein levels and the induction of apoptosis by endogenous NO was observed in macrophages. In contrast, a stable level of IAP protein in mesangial cells might represent a mechanism for the resistance of the cells to endogenously produced NO.
