Parotid acinar cells secrete proteins via two pathways that are distinct from salivary gland exocytosis: a constitutive-like pathway, insensitive to secretory stimulation and operating continuously, and a minor regulated pathway, which responds to low level stimulation. These two secretory pathways are hypothesized to provide the continual and neurally modulatable components of basal salivary secretion. The minor pathway has also been suggested to augment salivary output by priming secretory granule exocytosis. Long term goals of this project are to trace these pathways and understand their regulation and function at a molecular level using rat parotid glands as an experimental system. The first specific aim is to identify the carriers of the minor regulated pathway by a combination of techniques including biosynthetic labeling, subcellular fractionation, and immunofluorescence microscopy. The second specific aim is to develop a permeabilized cell system that faithfully reiterates secretion by the minor regulated pathway and by granule exocytosis. This system will be used to analyze the function of the minor regulated pathway and its role in priming for granule release.
The final aim of this project addresses a novel aspect of stimulus-secretion coupling in salivary acinar cells. The goal is to determine if stimulus-evoked relocation of the protein SNAP-23 from the basolateral membrane to the apical secretory apparatus serves to link cellular stimulation to exocytosis, and if so, how. In this aim, relocation will be examined further using parotid lobules and permeabilized acinar cells in experiments that include immunofluorescence and immunoelectron microscopy, antibody perturbation, and analysis of molecular interactions. Oral/salivary disorders such as xerostomia and ductal obstruction are primary or secondary consequences of impaired salivary secretion that may emphasize dysfunction in both basal and stimulated secretion. The new directions being taken in specifically addressing basal secretion and in examining what is potentially a key event in stimulus-secretion coupling may help to uncover the defects that cause the salivary dysfunctions. The findings may be broadly applicable in exocrine secretory cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE008941-14
Application #
6516436
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Program Officer
Kousvelari, Eleni
Project Start
1989-04-01
Project End
2004-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
14
Fiscal Year
2002
Total Cost
$308,529
Indirect Cost
Name
University of Virginia
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Castle, Anna M; Huang, Amy Y; Castle, J David (2002) The minor regulated pathway, a rapid component of salivary secretion, may provide docking/fusion sites for granule exocytosis at the apical surface of acinar cells. J Cell Sci 115:2963-73
Huang, A Y; Castle, A M; Hinton, B T et al. (2001) Resting (basal) secretion of proteins is provided by the minor regulated and constitutive-like pathways and not granule exocytosis in parotid acinar cells. J Biol Chem 276:22296-306
Castle, A M; Huang, A Y; Castle, J D (1998) Immunoglobulin-derived polypeptides enter the regulated secretory pathway in AtT-20 cells. FEBS Lett 439:341-5
Arvan, P; Castle, D (1998) Sorting and storage during secretory granule biogenesis: looking backward and looking forward. Biochem J 332 ( Pt 3):593-610
Castle, A M; Castle, J D (1998) Enhanced glycosylation and sulfation of secretory proteoglycans is coupled to the expression of a basic secretory protein. Mol Biol Cell 9:575-83
Castle, A M; Huang, A Y; Castle, J D (1997) Passive sorting in maturing granules of AtT-20 cells: the entry and exit of salivary amylase and proline-rich protein. J Cell Biol 138:45-54
Castle, J D; Castle, A M (1996) Two regulated secretory pathways for newly synthesized parotid salivary proteins are distinguished by doses of secretagogues. J Cell Sci 109 ( Pt 10):2591-9
Castle, A M; Schwarzbauer, J E; Wright, R L et al. (1995) Differential targeting of recombinant fibronectins in AtT-20 cells based on their efficiency of aggregation. J Cell Sci 108 ( Pt 12):3827-37
Girard, L R; Castle, A M; Hand, A R et al. (1993) Characterization of common salivary protein 1, a product of rat submandibular, sublingual, and parotid glands. J Biol Chem 268:26592-601
Castle, J D; Castle, A M (1993) Sorting and secretion of salivary proteins. Crit Rev Oral Biol Med 4:393-8

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