The overall goals of our project are to determine the molecular basis of the biogenesis of regulated secretion in the developing pancreas. Our previous work has shown that 1 to 2 days prior to birth, acinar cells respond to secretagogue stimulation with elevations of intracellular second massagers levels but do not couple this event to exocytosis despite the observation that all of the organelles of the exocytic pathway are present. Regulated exocytosis in response to hormonal stimulation becomes functional approximately 24-48 hours after birth (secretogenesis). Our studies show that secretogenesis is correlated with movement of rab3D (a marker exclusively for zymogen secretogenesis is associated with a dramatic but transient appearance of rab4 (24-48 hour postnatally) in the apical cytoplasm of the cell where it colocalizes with the actin terminal web and not with membranes of the secretory or endocytic pathway. We postulate that the transient expression of rab4 is related to the maturation of an actin """"""""clamp"""""""" that may be a terminal event the cascade of events leading to the maturation of regulated exocytosis. We intend to pursue this system further in order to determine the factors responsible for secretogenesis.
Specific aims are (1) to characterize the physiological significance of rab3 and rab4 during secretogenesis with regard to state of phosphorylation, interaction with regulatory proteins (e.g., rab GDI and proteins that may be involved in actin binding), and to evaluate function in cell line, AR42J whose regulated secretory pathway can be hormonally induced and in which specific mRNAs for rab proteins are eliminated with anti-sense nucleotides; (2) to examine the expression of other small GTP-binding proteins and members of the SNARE family that may be developmentally regulated during secretogenesis; (3) to study secretogenesis in organ culture under defined conditions in order to determine the effect of candidate hormones on maturation of regulated secretion; and (4) to examine an acinar cell tumor and a derived inducible cell line, AR42J, that exhibits many of the properties of normal prenatal pancreas for expression and distribution of rab proteins involved in normal secretogenesis. Comparison between the developing gland and the acinar cell tumor may provide information on the potential defects in secretion that characterize the morphology of this rat tumor and several types of human pancreatic cancer.
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