Characterization of receptor redistribution and regulatory volume decrease in rabbit alveolar macrophages

The present study was designed to examine the hypothesis that trasnporter/receptor recycling rates were involved in regulatory volume decrease (RVD). The alveolar macrophage is a useful tool for the study of membrane component recycling because it expresses a number of different rceptors including those for differric transferring, aMacroglobulin-protease complex, and mannose-terminated glycoproteins. The cellular distribution of these receptors depends on both the rate of internalization and the rate of exteriorization. Alterations in the rate of either limb"" of the recycling pathway between the cell surface and the endocytic apparatus can lead to an alteration in the distribution of receptors. Using recycling receptors as a model for membrane redistribution, involvement of membrane recycling in the regulation of ion transport during RVD was investigated. Hypo-osmotic incubation of cells as 37 ° C resulted in a rapid and reversible increase in surface receptor number for these three ligands. This increase was time, temperature, and dilution dependent. Examination of both the rates of internalization and exteriorization of receptors revealed that the increase in surface receptor number was due to a transitory decrease in the rate of receptor internalization without altering the rate of receptor exteriorization. Hypo-osmotic incubation of cells inhibited internalization of both occupied and unoccupied receptors. Both the rate of receptor internalization and surface receptor number returned to near control values after 30 minutes in hypo-osmotic media. To investigate whether the inhibition of receptor internalization represented a general inhibition of cellular endocytic processes, the effects of hypo-osmotic incubation on fluid phase pinocytosis were examined. HRP uptake by cells in hypo-osmotic solutions was inhibited to the same magnitude, and with similar kinetics as receptor mediated endocytosis. However, unlike receptor internalization, fluid phase uptake did not recover to control vlaues after 30 minutes of hypo-osmotic inbuation. this result is explained by the proposed existance of independent pathways for fluid phase pinocytosis and receptor mediated endocytosis. These studies represent the first report of RVD in alveolar macrophages. The process of RVD in these cells isdependent on the loss of K+, Cl-, and osmotically obliged water. The characterization of ion loss by ion substitution experiments as well as pharmacological assays indicate that the ion pathways operating during RVD in theese cells are most similar to the independent cation and anion conductance pathways reported to operate in human peripheral blood lymphocytes and platelets. Kinetic analysis of cation loss demonstrated that cation loss was rapid and preceded both the changes in surface receptor number and the initiation of RVD. The loss of K+ was first ordered and extrpolated back to the time of media dilution indicating that ion transporters were already present on the cell surface before receptor redistribution and were activated immediately upon dilution. Preliminary investigation into the cause of inhibition of receptor internalization indicate that although cell volume/morphology changes correlate with changes in surface receptor number, the changes induced by forces of swelling may not be the direct cause of inhibition. Alterations of intracellular pH as a result of hypo-osmotic incubation may be involved in the inhibition of internalization processes. Further investigations are required to determine this. Hypo-osmotic incubation of cells provides a unique ""tool"" for further study into the mechanism of recycling and membrane movement. It is the first reported procedure that affects the internalization ""limb"" of the recycling pathway without affecting the exteriorization ""limb."" In addition, hypo-osmotic incubation provides an experimental means of separating the processes of fluid phase pinocytosis and receptor mediated endocytosis.""