Biochemical evidence for transcytotic absorption of polyaspartamide from the rat lung: effects of temperature and metabolic inhibitors.

ABSTRACT

Airway-to-perfusate polyhydroxyethylaspartamide (PHEA) absorption was studied in the isolated perfused rat lung at a reduced temperature and by the use of metabolic inhibitors, to kinetically clarify the mechanisms and cellular pathways of its active absorption. Fluorophore-labeled PHEA (F-PHEA; 7.4 kDa) was administered into the airways, and its absorption followed with time at 25 degrees C and in the presence of 2,4-dinitrophenol (DNP), ouabain (OUA), monensin (MON), and nocodazole (NOC). Across-dose absorption profiles were analyzed using a kinetic model incorporating active (V(max,P) and K(m,P)) and passive (k(a,P)) absorption from the pulmonary lung region alongside the competing, pulmonary-to-bronchial mucociliary escalator (k(E)). The model was validated at 25 degrees C and a lack of perturbation on the k(a,P) and k(E) values for passively absorbed solutes confirmed by studying the disposition of sodium fluorescein and 4.4 kDa fluorescein isothiocyanate-labeled dextran. F-PHEA absorption was significantly suppressed at 25 degrees C, compared with 37 degrees C, because of a significant decrease in the value of the maximum rate of active absorption, V(max,P) (4.37 --> 0.67 microg/min; p < 0.05), whereas the carrier-affinity term, K(m,P), was statistically unchanged. F-PHEA's active absorption was also significantly inhibited by DNP (> or =0.5 mM), OUA (> or =50 microM), MON (> or =10 microM), and NOC (> or =1 microM), whereas these inhibitors had no significant effect on the values for k(a,P) and k(E). Thus, F-PHEA's pulmonary active absorption in the rat lung was temperature- and adenosine 5'-triphosphate-derived intracellular energy-dependent (DNP and OUA inhibition) and apparently mediated via transcytosis through cytoplasmic endosomes and microtubules (MON and NOC inhibition).