ENERGY AND CYTOSKELETAL REQUIREMENTS FOR MILK SYNTHESIS AND SECRETION BY ACINI FROM RAT MAMMARY GLAND

JOHN JOSEPH SMITH, Purdue University

Abstract

Incubation of acini (aveoli) from rat mammary gland with metabolic and cytoskeletal inhibitors produced a variety of effects on cell function. Cell viability was monitored during incubation by measurement of lactate dehydrogenase (LDH) activity in media and by light and electron microscopic morphological examination. Based on immunoprecipitation, caseins and whey proteins were secreted by acini. Addition of iodoacetate, 2,4-dinitrophenol, cyanide, cycloheximide, or cytochalasin B inhibited both synthesis and secretion proteins. Colchicine, however, specifically inhibited protein secretion. These results were confirmed by dose response and time course studies. Characteristic ultrastructural changes were produced by each inhibitor. Uptake of radiolabeled amino isobutyric acid (AIB), a nonmetabolizable amino acid, was reduced after incubation with all inhibitors except iodoacetate and 2,4-dinitrophenol, which produced no effect. Incorporation of tritiated uridine into trichloroacetic acid insoluble extracts of acini was inhibited by colchicine, vinblastine, cytochalasin B and, at high concentrations, by 2,4-dinitrophenol; cyanide and cycloheximide stimulated uridine incorporation. Intracellular protein transport as determined by measuring tritiated leucine incorporation into rough endoplasmic reticulum, Golgi apparatus, and secretory vesicles was variously effected during incubation with metabolic and cytoskeletal inhibitors. Cycloheximide, iodoacetate, 2,4-dinitrophenol, and cytochalasin B appeared to block protein synthesis on ribosomes of rough endoplasmic reticulum, whereas cyanide caused accumulation of radiolabelled proteins in secretory vesicles. Vinblasine inhibited transport of protein from rough endoplasmic reticulum to Golgi apparatus and colchicine appeared to cause accumulation of protein in all three cell fractions. Iodoacetate reduced acinar lactose content but was without effect on lactose synthetase activity. Cyanide, cycloheximide, and vinblastine reduced lactose synthetase activity but had no effect on tissue lactose concentration. Cytochalasin B reduced acinar glucose incorporation but was without effect on lactose content or lactose synthetase activity. Colchicine and 2,4-dinitrophenol did not alter glucose incorporation, lactose content, or lactose synthetase activity. Lactose secretion, however, was reduced by all inhibitors examined. Results of these studies indicate that sustained protein secretion depends on continued protein synthesis, which is an energy dependent process. Lactose secretion appeared to be linked to protein secretion. Results obtained from studies with colchicine further support the proposed role for microtubules in milk secretion.

Degree

Ph.D.

Subject Area

Livestock

COinS