Endotoxin induces PAF production in the rat ileum: Quantitation of tissue PAF by an improved method

PAF (platelet-activating factor) is an endogenous mediator of endotoxin (LPS) shock and intestinal injury. In the present study we used an improved method to quantitate intestinal PAF after LPS injection. Both column and thin layer chromatography (TLC) were used to purify PAF. We found that using C18 column eluted sequentially with 10% acetic acid, ethyl acetate and 70% ethanol, yielded consitent results. TLC yielded falsely high PAF values, possibly from an unknown tissue lipid which co-migrated with PAF, or from toxic ingredients in the silica gel. Moreover, addition of optimal amounts of Tween-20 or ethanol in the bioassay samples enhanced PAF solubility and markedly improved PAF recovery. Lastly, dilution and heparinization of platelet-rich plasma greatly improved the sensitivity of the bioassay. The overall PAF recovery under these optimal conditions was 70–80%. We found that LPS (2–10 mg/kg, iv, 90 min) stimulated PAF production in the rat ileum, but not in the jejunum and colon. The difference in PAF production did not correlate to the numbers of sequestered neutrophils (reflected by myeloperoxidase levels) after LPS injection. This selective PAF production may account for the special vulnerability of the ileum to develop injury during endotoxemia.     

Thermodynamic analysis of active sodium transport and oxidative metabolism in toad urinary bladder.

Measurements of electrical current and oxygen consumption were carried out concurrently under voltage clamp conditions in 11 toad hemibladders. Inhibition of active transport with amiloride then permitted evaluation of the passive conductance and the rate of basal oxygen consumption Jbr, allowing the simultaneous determination of the rates of active sodium transport JaNa and suprabasal oxygen consumption Jsbr-JaNa and Jabr were linear functions of the electrical potential difference over a range of +/- 80 mV. This allowed the comprehensive application of a linear nonequilibrium thermodynamic formalism, leading to the evaluation of the affinity A (negative free energy) of the metabolic reaction driving transport, all phenomenological coefficients, and the degree of coupling q relating transport to metabolism. Values of A determined by two techniques were A1=56.0 +/- 5.8 and A2=58.2 +/- 6.5 kcal per mole. Values of q determined by two techniques agreed well and were less than 1, indicating incompleteness of coupling, and hence lack of fixed stoichiometry between Na transort and O2 consumption. The affinity and the electromotive force of sodium transport ENa are not closely correlated, reflecting the fact that ENa comprises both kinetic and energetic factors.