The uptake of 45Ca2+ was studied in microsomes prepared from isolated mouse pancreatic acini. These microsomes accumulated 45Ca2+ in the presence of ATP; uptake was potentiated by addition of oxalate. Sequestered microsomal 45Ca2+ was only gradually removed by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) but was readily released by the divalent cation ionophore A23187. Inhibitors of mitochondrial oxidation and mitochondrial calcium transport had little effect on microsomal 45Ca2+ uptake. A separate subcellular fraction enriched in plasma membranes took up 45Ca2+ poorly compared with the microsomal fraction. Half-maximal 45Ca2+ uptake by the microsomal fraction was observed at a free Ca2+ concentration of 1.1 microM. 45Ca2+ uptake was dependent on Mg-ATP and showed a pH optimum at 6.8-7.0. Subfractionation of the total microsomes into "heavy" and "light" microsomal fractions indicated higher 45Ca2+ uptake activity associated with the heavy fraction. A Ca2+-activated, Mg2+-dependent ATPase was demonstrated in this fraction. Stimulation of pancreatic acini with the cholecystokinin analogue caerulein prior to homogenization increased the subsequent rate of 45Ca2+ uptake by the microsomal fraction.
SummaryTumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including alcoholic liver disease. In the liver, Kupffer cells are the primary source of the cytokine. Obliteration of Kupffer cells or neutralization of TNF-α by anti-TNF-α antibody or by an antisense oligonucleotide prevents ethanol-mediated liver damage. In this study, we report the identification of yet another highly efficacious gene-silencing molecule, the short interfering RNA (siRNA), SSL3, against TNF-α. The efficacies of various siRNA duplexes were tested against TNF-α mRNA in primary cultures of rat Kupffer cells. SSL3 (25 nM) inhibited lipopolysaccharide (LPS)-induced secretion of TNF-α by 55% (p<0.005) with a proportionate reduction in TNF-α mRNA; the inhibitory effect lasted for at least 96 h. Four nucleotide mismatches to SSL3 completely abolished the inhibitory effects of SSL3, suggesting the sequence specificity of the siRNA. Further, the in vivo efficacy of SSL3 was assessed following the i.v. administration of two doses (140 μg/kg body weight/day for two days) of liposome-encapsulated SSL3. The LPSinduced TNF-α secretion was inhibited by > 60% (p < 0.05) by SSL3 pre-treatment. These data demonstrate the identification of an siRNA against TNF-α, which, as a liposomal formulation, has therapeutic potential in the treatment of inflammatory diseases mediated by TNF-α.
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