Endotoxin binding on capillary endothelium and myocyte plasma membranes in perfused rat heart

Abstract

This work uses a novel heart-perfusion technique to measure [³H]-lipopolysaccharide ([³H]-LPS) binding on capillary endothelium and myocyte cell membranes in Sprague-Dawley rats. Free or serum-containing Ringer-Lock buffer was infused at a rate of 1 ml/min and in the presence of 20 mM K⁺ and [ ³H]-LPS through an aortic cannula, and the effluent was collected through a catheter introduced into the right atrium cavity. The capillary endothelial lining was removed by CHAPS treatment to expose the cardiac myocyte surface. A physical model describing 1:1 binding stoichiometry of [³H]-LPS with its receptors is proposed and the mathematical equations derived allow for the calculation of binding constants (k_n), reversal constants (k_-n), dissociation constants (k_d), and residency time constants (τ). The results showed that the presence of serum in the perfusate, slowed the binding of [³H]-LPS with the endothelial lining and myocytes, but increased the residency time by 3- and 50-fold, respectively. Hence, the endothelium and myofiber may contain LPS receptors that can bind more strongly with the ligand in association with sCD14-like and LBP-like molecules in rat serum. Thus it is postulated that the affinity of LPS to its receptor subtypes is not strictly and specifically dependent on the CD14 binding profile.