A NOVEL METHOD FOR THE STUDY OF AUTOPHAGY - DESTRUCTION OF HEPATOCYTIC LYSOSOMES, BUT NOT AUTOPHAGOSOMES, BY THE PHOTOSENSITIZING PORPHYRINTETRA(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 hep atocytic lysosomes. A major fraction of the lysosomal enzymes acid pho sphatase and N-acetyl-beta-D-glucosaminidase was inactivated by TPPS4 after 20 h of contact with the drug in vivo in the absence of photoact ivation. 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 cytos olic enzyme that accumulated in lysosomes as a result of autophagy dur ing a 2 h incubation of hepatocytes at 37 degrees C in the dark (in th e presence of the proteinase inhibitor leupeptin to prevent degradatio n of intralysosomal LDH). Phoroactivation of TPPS4 also induced lysoso mal rupture, with a loss of lysosomal enzymes, autophagocytosed LDH, e ndocytosed I-125-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 af fected by TPPS4. TPPS4 may thus be useful as a selective lysosomal (or endosomal) perturbant in the study of autophagic-endocytic-lysosomal interactions.