Studies on the mechanism of swelling-induced lysosomal alkalinization in vascular smooth muscle cells

Abstract

Previous studies in renal cells and hepatocytes have shown that cell swelling leads to a rapid and reversible increase in pH in acidic cellular compartments, including lysosomes. Among the consequences are an inhibition of proteolysis. The present study shows that a similar lysosomal alkalinization occurs upon osmotic swelling of vascular smooth muscle cells, as evidenced by acridine orange and fluorescein isothiocyanate fluorescence. Furthermore, we have studied the mechanism underlying lysosomal alkalinization, which had remained unclear. The lysosomal alkalinization was not abolished by inhibition of vacuolar H⁺-ATPases (100 nM bafilomycin), Cl⁻ channels [100 μM] 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), carbonic anhydrase (100 μM acetazolamide) or Na⁺/H⁺ exchange (10 μM HOE 694). The Ca²⁺ ionophore A23187 (10 μM) led to a slight increase in lysosomal pH, but removal of extracellular Ca²⁺ and depletion of cellular Ca²⁺ stores (100 nM thapsigargin) did not appreciably blunt the swelling-induced lysosomal alkalinization. In the presence of bafilomycin the alkalinizing effect of osmotic cell swelling was not reversible, in contrast to that of NH₄Cl. In conclusion, osmotic swelling of vascular smooth muscle cells leads to lysosomal alkalinization, presumably in large part through activation of a hydrogen ion leak.