Cyclic AMP potentiates substance P-induced amylase secretion by augmenting the effect of calcium in the rat parotid acinar cells

To establish a continuous cell line, freshly prepared rat parotid acinar cells were stably transfected with a plasmid vector containing the SV40 large T antigen. The acinar origin of these cells was confirmed by Western blotting, enzyme analysis, and morphological analysis. Transformed cells grown in 10% rat serum showed a modest reduction in cell number after 7 days and a concentration- and time-dependent increase in amylase levels ∼16 times greater than those observed in fetal bovine serum-treated cells. Ultrastructural analysis revealed that cells grown in rat serum harbored protein-filled secretory granules localized adjacent to the endoplasmic reticulum, and punctate amylase-specific immunofluorescence distributed throughout the cytoplasm was consistent with the presence of amylase in secretory organelles. Clonal cells express tissue-specific proline-rich proteins and the four protein kinase C isozymes present in primary culture. Carbachol and isoproterenol stimulated [3H]protein secretion and isoproterenol enhanced amylase secretion from cells grown in rat serum. Moreover, norepinephrine, carbachol, and substance P produced a time- and concentration-dependent rise in cytoplasmic Ca2+. This continuous cell line of parotid acinar cells, which after treatment with rat serum retains the basic structural and functional properties of primary culture cells, will be utilized as a model system for studying long-term biological processes that regulate parotid cell function.

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Regulation of phosphatidate synthesis by secretagogues in parotid acinar cells

Biochemical Journal ◽

10.1042/bj2040587 ◽

1982 ◽

Vol 204 (2) ◽

pp. 587-592 ◽

Cited By ~ 42

Author(s):

S J Weiss ◽

J S McKinney ◽

J W Putney

Keyword(s):

Steady State ◽

Substance P ◽

Specific Radioactivity ◽

Acinar Cells ◽

Half Time ◽

Muscarinic Antagonist ◽

Parotid Acinar Cells ◽

Rat Parotid ◽

In Cells

The metabolism of phosphatidate in rat parotid acinar cells was investigated, particularly with regard to the actions of agonists known to act by mobilizing Ca2+. When cells were incubated in medium containing 10 microM-[32P]Pi, phosphatidate was rapidly labelled, approaching an apparent steady-state with a half-time of approx. 20 min. Methacholine provoked a more than doubling of phosphatidate radioactivity, which was reversed by the muscarinic antagonist atropine. These results suggest that phosphatidate labels to near steady-state rapidly and that in cells prelabelled for 60 min the increase in radioactivity induced by agonists probably reflects net synthesis rather than an increase in specific radioactivity. Phosphatidate synthesis in response to methacholine was rapid and occurred, within the resolution of a few seconds, with no measurable latency. Adrenaline and substance P also stimulated phosphatidate synthesis but both agonists were less efficacious than methacholine. A Ca2+ ionophore, ionomycin, did not provoke phosphatidate synthesis. By using a protocol that eliminates the receptor-regulated Ca2+ pool, it was demonstrated that methacholine-induced phosphatidate formation does not come about as a consequence of Ca2+ influx nor of Ca2+ release. These results indicate that the phosphatidate synthesis response has characteristics compatible with its previously suggested role as a primary mediator of membrane Ca2+-gating.

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