Mechanisms of lysosomal enzyme release from leukocytes exposed to immune complexes and other particles.

Human PMN release lysosomal enzymes (beta-glucuronidase, acid phosphatase) when exposed to immune complexes, but do not release cytoplasmic LDH. The cells remain viable, and failure of LDH to appear in supernatants is not due to selective absorption or inactivation. Release of enzymes is not due to platelet contamination and is only partially enhanced by fresh serum. The selective release of lysosomal enzymes after uptake of complexes resembles that induced by inert particles of zymosan, and can be distinguished from the concurrent release of all enzymes after cell death induced by membrane-lytic crystals of MSU. Uptake of complexes, zymosan, or MSU particles is accompanied by concomitant increases in C-1 oxidation of glucose. Although MSU-induced damage can be retarded by the presence of Tris buffer, immune complexes and zymosan selectively release lysosomal hydrolases in the presence or absence of Tris buffer. Agents which elevate the level, within cells, of cAMP (PGE(1), theophylline, 2-CA) and cAMP itself inhibit the selective extrusion of acid hydrolases from leukocytes without affecting the viability of cells. Leukocytes may respond to immune particles by regurgitating a portion of their lysosomal hydrolases during phagocytosis.

Heat inhibition of reticulocyte protein synthesis. Evidence for a mechanism independent of the hemin-controlled repressor.

Incubation of rabbit reticulocytes at 45 degrees C results in a prompt but reversible decrease in protein synthesis and a concomitant conversion of polyribosomes to smaller aggregates. These effects occur even in the presence of 100 micrometer hemin in the incubation medium. There is also inhibition of heme synthesis but this occurs at a later time than the effect on protein synthesis. The inhibtion of heme synthesis results from a decrease in activity of beta-aminolevulinic acid synthetase. This decrease of heme synthesis appears to be secondary to the inhibition of protein synthesis with resultant accumulation of intramitochondrial heme (which will decrease beta-aminolevulinic acid synthetase activity). An inhibitor of reticulocyte cell-free protein synthesis formed in the postribosomal supernatants of cells incubated at both 45 and 37 degrees C but not at 0 degrees C. No temporal or quantitative differences in the amount of this inhibitor from cells treated at either 37 or 45 degrees C was apparent. The inhibitor was not found in the fraction where the hemin-controlled repressor is isolated. It is concluded that heat inactivation of intact reticulocyte protein synthesis does not depend upon a decrease in heme synthesis, heme concentration or generation of the hemin-controlled repressor. Furthermore, it appears that the inhibitor formed in the post-ribosomal supernatant cannot be the sole cause of the heat inhibition of protein synthesis.

Ethanol inhibition of rabbit reticulocyte haem synthesis at the level of delta-aminolaevulinic acid synthetase.

Ethanol inhibition of rabbit reticulocyte synthesis occurs as a result of a decrease in haem synthesis. The present study therefore was undertaken in order to localize the inhibitory site of ethanol on the haem biosynthetic pathway. Ethanol (0.05--0.15 M) inhibition of reticulocyte protein synthesis was prevented by simultaneous incubation with 0.025--1 mM delta-aminolaevulinic acid (ALA). Ethanol inhibited both 14C-glycine and 14C-ALA incorporation into haem. However, the extent of haem formation with 14C-ALA as substrate in the presence of ethanol was still equal to that when 14C-glycine was used. These data suggest that ethanol inhibits the haem synthetic pathway at several loci, but that the decrease in haem synthesis, responsible for the decrease in protein synthesis, is due to the inhibition at the rate-limiting enzyme, delta-aminolaevulinic acid synthetase (ALA-S). To confirm this, ALA-S activity was then directly measured in intact reticulocytes, and it was shown that ethanol indeed inhibited its activity. The inhibition of ALA-S was prevented by 10(-4) M dibutyryl cyclic AMP (db cAMP) or theophylline, agents which elevate intracellular cAMP and which have previously been shown to prevent and reverse ethanol inhibition of haem and protein synthesis. Thus, it appears that cAMP protects against ethanol toxicity by preventing inhibition of ALA-S.