Isolated Parotid Cells Research Articles

Development of salivary gland cell lines for studies of signaling and physiology.

We developed and characterized an immortalized rat parotid cell line to use in salivary gland studies. The cells were immortalized by retroviral transduction of SV40 large T antigen into isolated parotid cells. Using immunocytochemical techniques, we found that the immortalized epithelial cells were ductal, rather than acinar, in nature. Cells were grown under coculture conditions with lethally irradiated NIH3T3 cells. One cell line, which was designated RPG1/SV40 cells (for rat parotid gland 1/SV40 transformant), was selected for characterization. These cells formed a sheet epithelium with tight junctions and a measurable transepithelial resistance. RPG1/SV40 cells responded to muscarinic receptor (carbachol) and/or P2 purinoceptor (ATP and UTP) stimuli with increases in the following: (1) intracellular free-calcium concentration ([Ca2+]i); (2) the short-circuit current (ISC) across the epithelium; (3) the tyrosine phosphorylation of PKC delta; and (4) MAP kinase activity. Thus, the cells appear to be useful for a wide range of studies involving physiology, biochemistry, and signal transduction approaches.

Esterification of D-mannoheptulose confers to the heptose inhibitory action on D-glucose metabolism in parotid cells.

D-mannoheptulose, but not its hexaacetate ester, inhibits, in a competitive manner, D-glucose phosphorylation by either purified beef heart hexokinase or crude parotid gland homogenates. Yet, D-mannoheptulose hexaacetate, but neither the unesterified heptose nor acetate or its methyl ester, inhibits D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to 14CO2 and 14C-labelled acidic metabolites and amino acids in intact isolated parotid cells. It is proposed, therefore, that D-mannoheptulose hexaacetate crosses efficiently the plasma membrane of parotid cells and, after intracellular hydrolysis, allows inhibition of D-glucose phosphorylation by the unesterified heptose. The ester of D-mannoheptulose could thus represent a useful tool to inhibit hexose phosphorylation and interfere with cell growth in cells otherwise resistant to the heptose.

Substance P stimulation of amylase release by isolated parotid cells and inhibition of substance P induction of salivation by vasoactive peptides

Substance P stimulation of salivation in rats has been studied as has its in vitro enhancement of amylase release by isolated parotid cells. The extent of the stimulation on amylase release by isolated parotid cells was dependent upon the concentration of substance P, with the minimum effective concentration being 1 nM. The substance P effect was detectable within 1 min after incubation and lasted for at least 50 min. Substance P stimulation was demonstrable at 25–37°C but not at 0°C. Adrenocorticotropic hormone (ACTH), thyrotropin-releasing hormone (TRH), vasopressin and neurotensin had no effect on amylase release. These results suggest that substance P may act directly on the parotid cells. Examination of the salivary-stimulating activity of fragments of substance P showed that the C-terminal octapeptide and (pyroglutamyl)hexapeptide were active, although less potent than substance P, whereas its free acid, C-terminal tetra- and tri-peptides were inactive. Vasopressin, angiotensin II and neurotensin could inhibit substance P induced salivation, whereas TRH, ACTH and somatostatin had no effect. Amylase activity per unit volume of saliva was not changed by the injection of vasopressin, angiotensin II or neurotensin. These vasoactive peptides did not affect substance P stimulation of amylase release by isolated parotid cells. The results indicate that vasopressin, angiotensin II and neurotensin inhibit the action of substance P on salivation at sites other than the parotid cells.