Characterization of synthesis and storage of TGF-alpha in rat parotid acinar and intercalated duct cells

Abstract

Although the expression and biological role of transforming growth factor-alpha (TGF-alpha) have been explored in a variety of normal cells in mammalian species, little is known about the storage of TGF-alpha in secretory cells of exocrine organs. Parotid glands from four rats were homogenized for RNA isolation followed by reverse transcription-polymerase chain reaction to determine the presence of TGF-alpha message. In situ hybridization using a hamster-specific TGF-alpha riboprobe was done on paraffin sections. Parotid gland and isolated acinar cells were processed for transmission electron microscopy (TEM) and postembedding immunogold labeled for TGF-alpha. Gold particles were counted on approximately 200 granules in 10 acinar cells and in 10 intercalated duct cells. Labeling density was calculated as the number of gold particles per square micrometer +/- SD. Statistical significance was calculated using one-way analysis of variance. Using multiple technologies, we have established that rat parotid acinar and intercalated duct cells synthesize TGF-alpha and store the precursor form of this cytokine in their secretory granules.

Age-related Alterations in IL-1β, TNF-α, and IL-6 Concentrations in Parotid Acinar Cells from BALB/c and Non-obese Diabetic Mice

“…Serous acini are the sole secretory cell type in parotid gland (Tandler 1978) and are an important cell type in most exocrine glands. The secretory function of serous acini can be easily determined using assays of amylase and LDH release (Login et al 1997). In addition, parotid glands affected by Sjögren's syndrome show a chronic progressive decrease in function in humans (Wise et al 1988) and in NOD mice (Hu et al 1992).…”

“…RT-PCR detection of CD3 , F4/80, CD20, and G3PDH mRNAs was done using mouse-specific primers as previously described (Login et al 1997), slightly modified. In brief, thirty cycles for G3PDH, CD3 , and CD20 and 35 cycles for F4/80 were performed using a Perkin Elmer Cetus GeneAmp PCR System 2400 (Foster City, CA) with the following temperature profiles for G3PDH and F4/80 at 94C for 1 min, 60C for 1 min, and 72C for 1 min; for CD3 at 94C for 30 sec, 56C for 30 sec, and 72C for 1 min; and for CD20 at 94C for 1 min, 55C for 1 min, and 72C for 1 min.…”

“…Parotid glands and spleens were fixed in 10% neutral buffered formalin for 2 hr at 20C and processed routinely on a Tissue-Tek VIP processor (Miles; Naperville, IL), beginning with ethanol dehydration (Login et al 1997). Paraffin sections were cut at 4 m in preparation for staining by hematoxylin and eosin or for immunohistochemistry.…”

“…Using a Vectastain Elite ABC kit (Vector Laboratories; Burlingame, CA), 4-m paraffin sections of mouse parotid gland were stained for a cytoplasmic T-cell-specific antigen, CD3, with rabbit anti-human CD3 IgG (1:30, 1 hr, 20C) (Dako; Carpinteria, CA), secondary biotinylated swine anti-rabbit IgG (1:200, 30 min, 20C) (Dako), and avidin-horseradish peroxidase conjugate (1:400, 30 min, 20C) (Vector) (Login et al 1997). A murine macrophage membrane antigen, F4/ 80, was detected using rat anti-mouse F4/80 IgG (1:200, 1 hr, 20C) (Serotec; Raleigh, NC), secondary biotinylated rabbit anti-rat IgG (1:200, 30 min, 20C) (Vector), and avidinhorseradish peroxidase conjugate (1:125, 30 min, 20C) (Vector).…”

IL-1β and IL-6 in mouse parotid acinar cells: characterization of synthesis, storage, and release

“…RNA from freshly excised mouse parotid gland or dispersed parotid acini was also isolated. The RT-PCR reaction was performed as previously described (30). The efficiency of rat primer homology to mouse was determined by oligo primer analysis software (National Biosciences, Plymouth, MN).…”