Monocrotaline (MCT) is a pyrrolizidine alkaloid that causes pulmonary hypertension in rats by mechanisms which remain largely unknown. MCT is thought to be activated in the liver to a reactive intermediate that is transported to the lung where it causes endothelial injury. Our previous pharmacokinetic work demonstrated significant sequestration of radioactivity in red blood cells (RBCs) of rats treated with [14C]MCT. To determine whether this RBC sequestration might be important in the transport of reactive MCT metabolites, we compared the effect of inclusion of RBCs in the perfusion buffer on the extent of covalent binding of [14C]MCT to rat lungs in tandem liver-lung preparations. The potential effect of RBCs in stabilizing reactive intermediates was evaluated by preperfusion of isolated liver preparations with [14C]MCT with and without RBCs, separation and washing of the RBC fraction, and subsequent (90 min later) perfusion of washed RBCs or buffer alone in isolated perfused lungs. Covalent binding to lung tissues was determined by exhaustive methanol/chloroform extractions of unbound label from homogenized lung tissue followed by scintillation counting of residual 14C. Covalent binding was expressed as picomole MCT molecular weight equivalents/mg protein. Comparison of the relative capability of these isolated organ preparations for conversion of MCT to polar metabolites was done by extraction and HPLC analysis of perfusate at the end of the experiment. Isolated livers converted 65-85% of MCT to polar metabolites compared with less than 5% conversion in the isolated lungs. Inclusion of RBCs in the buffer of tandem lung liver preparations perfused with 400 m [14C]MCT increased the covalent binding to the lung from 97 ± 25 (buffer alone) to 182 ± 36 (buffer + RBC) pmol/mg protein. At the end of these perfusions, RBCs contained 1552 ± 429 pmol/mg hemoglobin of which 333 ± 98 pmol/mg hemoglobin resisted exhaustive solvent extraction. After 90 min at room temperature, buffer with 400 μm [14C]MCT preperfused in isolated livers resulted in covalent binding to isolated perfused lung of 0.8 ± 0.4 pmol/mg protein while washed RBCs isolated from buffer of similar liver preperfusions preparations resulted in 53 ± 7 pmol/mg protein bound to lung. Control groups perfused with 400 μm [14C]MCT in buffer or buffer + RBCs through isolated lungs only resulted in covalent binding of 2 ± 1 or 1 ± 0.6 pmol/mg protein respectively. We conclude: (1) RBCs significantly augment the transport of lung reactive MCT metabolites from the liver to the lung, (2) The MCT-derived 14C sequestered in the RBCs is capable of covalent interaction with lung tissue, and (3) RBCs stabilize a reactive metabolite of MCT which originates in the liver.
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