Lysosomes and Cartilage Resorption in Organ Culture 



53 



dation by increased release of a lysosomal acid protease (Fell and Dingle, 1963), a 

 similar mechanism was suspected for the hyperoxia effect. 



Fig. 2 shows the growth of two groups of rudiments. One group of eight rudi- 

 ments was exposed to 20o/o oxygen while the paired rudiments were exposed to SSVo 

 oxygen. At two-day intervals the amounts 

 of acid protease and acid phosphatase 

 in the medium were measured. The 

 release of both of these lysosomal enzy- 

 mes was increased in the group exposed 

 to elevated oxygen. 



The lysosome, a cytoplasmic body 

 containing a variety of hydrolytic en- 

 zymes characterized by acid pH optima 

 and structure-linked latency, was de- 

 veloped as a biochemical concept by 

 DE DuvE et al. (1955), and de Duve 

 (1963). The morphology of these single 

 membrane limited structures has been de- 

 scribed by NoviKOFF (1963), and their 

 presence in all mammalian cells except 

 the erythrocyte and spermatocyte seems 

 likely (Dingle, personal communication). 

 Alteration of the liproprotein membrane 

 leads to activation of the enzymes, either 

 by escape of enzyme or entrance of 



substrate. Many agents have been shown to produce such alteration of the membrane: 

 acid pH, ultraviolet light, vitamin A, streptococcal endotoxin, antigen-antibody 

 reactions, etc. Measurements of the free/bound ratio of the lysosomal enzymes in the 

 rudiments showed that the fragility of the lysosomal membrane was increased in 

 hyperoxia. 



Since the membrane of the lysosome can be stabilized by Cortisol in physiological 

 doses (Weissmann and Dingle, 1962), it was added to the cultures at 0.1 ;.'/ml. or 

 0.01 y/m\. This steroid afforded complete protection from hyperoxia, by both 

 histological and biochemical criteria. 



In work with excess of vitamin A, Fell and Dingle (1963) have shown that 

 lysosomal acid protease is responsible for the degradation of matrix. Ali (1964) has 

 shown that this protease is competitively inhibited by epsilon-amino-n-caproic acid 

 (EACA), a synthetic amino acid which structurally resembles lysine. When EACA 

 was added to our medium at 40 ;'/ml. it, like Cortisol, protected the rudiments from 

 the effects of hyperoxia. 



It appeared evident that excess oxygen was activating the lysosomes of the 

 chondrocytes and releasing an acid protease which led to loss of matrix. What is the 

 mechanism for this effect — acid pH, change in cell membrane permeability, or a 

 direct effect on the lysosome? Kenny et al., (1959) suggested a blockage in the Krebs 

 cycle leading to accumulation of lactic and citric acids and a lowered pH as the 

 mechanism of bone resorption in hyperoxia. I have not found this to be the case in 

 our experiments. Citrate and lactate production, pH and glucose consumption are 



days in culture 



Fig. 2. Lengths of control .ind experimental rudiments 



shown in line graph. Release of acid phosphatase into 



medium shown in bar graph 



