Hydrodynamic properties of mucins secreted by primary cultures of guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution.

Abstract

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (Mw) for three different preparations ranged from 3.3 x 10(6) to 4.7 x 10(6) g/mol (corresponding to an average structure of 1-2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5 x 10(-8) to 6.4 x 10(-8) cm2/s were calculated using the Svedberg equation. A polydispersity index (Mz/Mw) of approximately 1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05 x 10(-8) cm2/s (inter-quartile range = 1.11 x 10(-7) to 6.08 x 10(-8) cm2/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98 x 10(-9) cm2/s (inter-quartile range = 1.47 x 10(-8) to 3.25 x 10(-9) cm2/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D20,w) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell.