Quantitative assessment of fibrinogen cross-linking by epsilon aminocaproic acid in patients with end-stage liver disease.

Analysis of the effectiveness of antifibrinolytic therapy for liver transplant recipients is hampered by lack of quantitative assays for assessing drug effects. We adapted chemical engineering tools used in polymerization studies to quantify fibrinogen cross-linking by plasma from liver transplant patients obtained before and after epsilon aminocaproic acid (EACA) therapy. A target fluorescein isothiocyanate-fibrinogen (FITC-fibrinogen) molecule was constructed; it fluoresces in a quantifiable pattern when in solution, and undergoes cross-linking in the presence of plasmin inhibitors. Cross-linking quenches the fluorescent signal, and the quenching is a quantifiable endpoint. Thus fluorescence from this reporter molecule can be used to assess functional improvement in fibrinogen cross-linking as a result of antifibrinolytic therapies, and it is sensitive to picomolar amounts of plasmin inhibitors and activators. Cross-linking of FITC-fibrinogen by patient plasma, before and after EACA therapy, was assessed using fluorescence spectrometry. Fluorescence patterns from FITC-fibrinogen indicated no significant cross-linking of the target fibrinogen as a consequence of EACA in posttreatment plasma. When the fibrinogen-FITC target was assayed without plasma in the presence of EACA at concentrations that bracket therapeutic levels (100 and 400 microg/ml), significant fluorescence quenching (target FITC-fibrinogen cross-linking) was achieved. These results suggest that fibrinogen-FITC fluorescence is sensitive enough to detect EACA activity in clinically relevant ranges, but that EACA given in usual doses is insufficient to promote fibrinogen cross-linking in patients with end-stage liver disease.

Heterotrimeric G protein subunits are located on rat liver endosomes.

BACKGROUND: Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors. RESULTS: By Western blotting Gsalpha, Gialpha1,2, Gialpha3 and Gbeta were enriched in both canalicular (CM) and basolateral (BLM) membranes but also readily detectable on three types of purified rat liver endosomes in the order recycling receptor compartment (RRC) > compartment for uncoupling of receptor and ligand (CURL) > multivesicular bodies (MVB) >> purified secondary lysosomes. Western blotting with antibodies to Na, K-ATPase and to other proteins associated with plasma membranes and intracellular organelles indicated this was not due to contamination of endosome preparations by CM or BLM. Adenylate cyclase (AC) was also identified on purified CM, BLM, RRC, CURL and MVB. Percoll gradient fractionation of liver postnuclear supernatants demonstrated co-occurrence of endosomes and heterotrimeric G protein subunits in fractions with little plasma membrane markers. By confocal microscopy, punctate staining for Gsalpha, Gialpha3 and Gbeta corresponded to punctate areas of endocytosed Texas red-dextran in hepatocytes from control and cholera toxin-treated livers. CONCLUSION: We conclude that heterotrimeric G protein subunits as well as AC likely traffic into hepatocytes on endosome membranes, possibly generating downstream signals spatially separate from signalling generated at the plasma membrane, analogous to the role(s) of internalized insulin receptors.

Effect of animal bedding on rat liver endosome acidification.

Animal beddings, such as pine products, and environmental factors are known to induce liver drug-metabolizing cytochrome P450 enzymes. We observed that a change to pine-based rat bedding altered baseline and cAMP-stimulated rates of acidification in rat liver endosomes, apparently by decreasing ATP-dependent proton transport in the presence and absence of chloride. Although cAMP altered phosphorylation of protein kinase B and extracellular signal-regulated kinases 1 and 2 (ERK 1,2) and p38 mitogen-activated protein kinases, changes in housing conditions did not affect baseline or cAMP-stimulated values of these or other selected signaling molecules. We conclude that compounds in rat bedding may alter not only drug metabolism, but also aspects of endocytosis.

Hyperacidification of cellubrevin endocytic compartments and defective endosomal recycling in cystic fibrosis respiratory epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments.