Factors influencing the in vitro production of a myocardial depressant factor

The mechanisms of the intrapancreatic formation of a myocardial depressant factor (MDF) were investigated in incubated pancreatic homogenates and in subcellular fractions. An incubation period of 90-120 min was found to be optimal for MDF production. The optimal pH for MDF production in pancreatic homogenates was found to be 5-6. MDF production required the presence of salts, but was not influenced by an anaerobic environment. Large concentrations of trypsin (i.e., 1-2 mg/ml) but not of chymotrypsin, lipase, or phospholipase A, inhibited MDF formation. Differential centrifugation of homogenates yielded several fractions which formed MDF upon incubation. Measurement of marker enzymes revealed that fractions containing lysosomes and zymogen granules produced MDF whereas fractions containing predominantly either mitochondria or microsomes did not. Moreover, addition of a sulfhydryl blocking agent (e.g., PCMB) to incubated subcellular fractions and homogenates prevented MDF formation. These data implicate the proteases of lysosomes and zymogen granules as enzymes which participate in the formation of MDF, although the precise mechanism is not completely known.