A novel method for the study of autophagy: destruction of hepatocytic lysosomes, but not autophagosomes, by the photosensitizing porphyrin tetra(4-sulphonatophenyl)porphine

A photoactivatable porphyrin, tetra(4-sulphonatophenyl)porphine (TPPS₄), was shown to accumulate in rat hepatocytes as a linear function of dose after intravenous injection, and to localize predominantly in hepatocytic lysosomes. A major fraction of the lysosomal enzymes acid phosphatase and N-acetyl-β-d-glucosaminidase was inactivated by TPPS₄ after 20 h of contact with the drug in vivo in the absence of photoactivation. On exposure of isolated hepatocytes to light, photoactivated TPPS₄ caused additional inactivation of the lysosomal enzymes as well as inactivation of intralysosomal lactate dehydrogenase (LDH), a cytosolic enzyme that accumulated in lysosomes as a result of autophagy during a 2 h incubation of hepatocytes at 37 °C in the dark (in the presence of the proteinase inhibitor leupeptin to prevent degradation of intralysosomal LDH). Photoactivation of TPPS₄ also induced lysosomal rupture, with a loss of lysosomal enzymes, autophagocytosed LDH, endocytosed ¹²⁵I-tyramine-cellobiose-asialo-orosomucoid and TPPS₄ from the lysosomes. However, LDH-containing autophagosomes, accumulated in the presence of vinblastine (a microtubule inhibitor used to prevent the fusion of lysosomes with autophagosomes or endosomes), were not affected by TPPS₄. TPPS₄ may thus be useful as a selective lysosomal (or endosomal) perturbant in the study of autophagic–endocytic–lysosomal interactions.