Radioautographic Analysis of the Secretory Process in the Parotid Acinar Cell of the Rabbit

Abstract

Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland . Rabbit parotids were chosen on the basis of size (2-2 .5 g per animal), ease of surgical removal, and amylase concentration . Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure . Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0 .175 M phosphate buffer (pH 7 .2) as primary fixative . Pulse labeling with leucine 3H, followed by a chase incubation, showed that the label is initially located (chase : 1-6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase : 16-36 min), condensing vacuoles (chase : 36-56 min), immature granules (chase : 56-116 min), and finally mature storage granules (chase : 116-356 min) . Distinguishing features of the parotid transport apparatus are : low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules . Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged . 290

Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions.

“…Immediately evident in this figure are the large variety of irregular granule shapes similar to the variety observed in intact cells (13), and the absence of recognizable mitochondria and microsomes present at higher incidence in secretion granule fractions obtained by repeated differential sedimentation. As demonstrated by Fig.…”

“…Pulse medium consisted of 10 ml of Krebs-Ringer bicarbonate medium (34) supplemented with unlabeled amino acids to one-half the final concentration found in Eagle's minimal essential medium and containing in addition 25 uCi/ml of reconstituted [14C]algal protein hydrolysate. At the end of the pulse, lobules were washed with 100 ml Medium FI2 (22) at 37~ and were then chase incubated in 100 ml FI2 for 4 h to allow the bulk of the incorporated radioactivity to be transported to secretory granules (13). The tissue in its entirety was then fractionated to obtain secretion granules according to the protocol outlined in the Results.…”

“…To prepare radioactively labeled parotid secretion granules, the tissue of four parotid glands was dissected into lobules (13) and pulse labeled in vitro for 30 min using a mixture of fifteen 14C-labeled amino acids. Pulse medium consisted of 10 ml of Krebs-Ringer bicarbonate medium (34) supplemented with unlabeled amino acids to one-half the final concentration found in Eagle's minimal essential medium and containing in addition 25 uCi/ml of reconstituted [14C]algal protein hydrolysate.…”

“…In our previous studies (13) we undertook a characterization by autoradiography of the secretory process of the acinar cell of the rabbit parotid gland in vitro to compare similarities and differences of the kinetics of intracellular transport of secretory protein from its site of synthesis on the rough endoplasmic reticulum to its site of storage in secretion granules in the parotid and in the guinea pig exocrine pancreas (28,29). The parotid should provide a system in which membrane lipolytic and proteolytic degradation is not expected.…”

“…Within the exocrine cell the population of accumulated granules, which can occupy as much as half of the cellular volume, is located in the apical cytoplasm between the Golgi region, where granules originate (2,13,28,29,54,55), and the apical cell membrane, where the period of storage is terminated by release of the granule contents into the extracellular space by the process of exocytosis (57).…”