Clonal strains of rat medullary thyroid carcinoma cells that produce neurotensin and calcitonin. Functional and morphologic studies.

Strains of rat medullary thyroid carcinoma cells were established by two different techniques which were designed to ensure that clonal populations originated from single cells. The clonal strains (44-3C1 and 6-23C6) secreted both immunoreactive calcitonin (CT) and neurotensin (NT) and had growth characteristics that were similar to those of the parent cell lines. Both the 44-3C1 strain and the parent 44-2 cell line consistently produced more CT and NT than the 6-23C6 clonal strain or the parent 6-23 cell line. Peptide secretion in these clonal strains was stimulated by calcium and norepinephrine. Both clonal strains, similar to the parent lines, produced 4 to 12 times more NT than CT. Immunohistochemical studies showed that all cells in both clonal strains stained positively for CT. NT was also present in all cells from both strains with approximately 2% of the cells showing intense staining for this peptide. Ultrastructurally, the cells contained membrane-bound secretory granules which had a mean diameter of 95 nm. Secretory granules were relatively numerous in only 2% of the cells where they tended to be concentrated in cell processes. These studies demonstrate that single rat medullary thyroid carcinoma cells produce both CT and NT and that these strains are useful models for studies of NT and CT biosynthesis and for investigations of the regulation of secretion of two peptides from a single cell type.

Regulation of neurotensin secretion in a mammalian C cell line: effect of dexamethasone.

We established in culture two colony clones of rMTC 44-2 cells, rMTC 44-2B and 44-2C which secrete substantially greater quantities of neurotensin (NT) than the parent cell line. We describe here the effects of the synthetic glucocorticoid, dexamethasone, on NT and cAMP release. Medium and intracellular levels of NT and cAMP were measured by specific RIAs. Long-term release experiments were performed in Dulbecco's Modified Eagle's Medium supplemented with 15% horse serum (DMEM). Short-term release experiments were performed in Krebs-Ringer-bicarbonate-glucose buffer (KRBG) supplemented with 1.0 mm Ca2+. Dexamethasone stimulated NT release and increased intracellular NT levels. The ED50 values for stimulation of NT release following 24 or 48 h incubation of cells in DMEM with dexamethasone were 5 X 10(-9) and 7 X 10(-9) M, respectively. Dexamethasone markedly enhanced intracellular levels of NT in rMTC 44-2 cells while it decreased cell growth. Cells pretreated with dexamethasone for 48 h released greater amounts of NT in response to Ca2+ (1.0 mM) with or without K+ (50 mM) or NE (10(-6) M) following a 10 min incubation with these substances in KRBG. This experimental paradigm was also used to measure the efflux of cAMP following a brief (10 min) exposure of cells to NE. We conclude that the rMTC 44-2B and 44-2C cells are useful tools for studying the effects of dexamethasone on the regulation of cell growth, as well as the secretion of NT and cAMP.

Regulation of neurotensin secretion in mammalian C cell lines: potassium and norepinephrine affect the rapid release of the peptide.

Calcitonin (CT) and neurotensin (NT) secreting cell lines (6-23,44-2) were established from a transplantable rat medullary thyroid carcinoma (rMTC). The 44-2 line was used to obtain two colony clones 44-2 B and C secreting 20-30-fold greater NT than CT. These cells were used to study the regulation of Ca2+-modulated NT secretion and to ascertain the role of K+ and NE in the rapid release of NT. Medium NT was measured by a specific RIA with the antiserum N-1-11. Secretion experiments were in replicate 35 mm dishes in Krebs-Ringer-bicarbonate buffer supplemented with 90 mg% glucose (KRBG). Ca2+ (0.5-4.0 mM) stimulated NT release in a dose-dependent manner with an ED50 of 2.0 mM. Ca2+ was required for NT release induced by the Ca2+ ionophore, ionomycin, also K+ (50 mM) and norepinephrine (NE). To determine the mode of NT release in the presence of control (1.0 mM Ca2+) or experimental conditions (Ca2+ 1.0 mM plus 10(-6) NE and/or 50 mM K+), 44-2 cells were incubated in KRBG using an experimental paradigm wherein medium was changed at 10-min intervals, and NT release was quantitated. NE stimulated release of NT by these cells and the amount of NT released with each repetitive pulse of NE remained constant throughout the experiment. K+ (50 mM) elicited a rapid release of NT in the first 0-10 min incubation and the amount of NT released into the buffer was greater than that measured with NE; however, in these experiments, the response to K+ declined progressively and reached basal values at 20-30 min. Our results show neither pulse stimulation nor continuous incubation of cells with NE affected the response of the cells to a subsequent challenge with K+. These results suggest the presence of differentially stimulated, releasable pools of NT in these cells. We conclude that these newly established 44-2 B and C cells provide a useful model to study the regulation of NT release.

Characterization of neurotensin production by a line of rat medullary thyroid carcinoma cells.

The rMTC 6-23 cell line was derived from a calcitonin-producing rat medullary thyroid carcinoma. During characterization of these cells we discovered that they also synthesize and secrete neurotensin (NT), a tridecapeptide originally isolated from the hypothalamus and not previously associated with C cells. Immunoreactive NT (iNT) was measured with two antisera: HC-8, which recognizes the COOH-terminal eight amino acids, and TG-1, which binds the NH2-terminal region of NT. By use of these antisera, 2 M acetic acid extracts of rMTC 6-23 cells were found to contain 0.7--5.0 pmol of iNT per mg of cell protein. Successive fractionation of iNT from cell extracts by gel filtration, ion-exchange chromatograpy, and reverse-phase high-pressure liquid chromatography showed at each step a peak of iNT that was indistinguishable from synthetic NT in its chromatographic behavior. Biologic activity of the iNT was confirmed by demonstrating the characteristic fall in arterial blood pressure in the rat after intravenous injection of material purified from rMTC 6-23 cells. Calcium (0.5--4.0 mM) stimulated release of iNT in a dose-dependent manner; the effect was maximal at 3--4 mM calcium. K+ (50 mM) stimulated release of iNT that was maximal in the presence of 1.0--1.5 mM calcium. Synthesis and secretion of iNT by these cells were shown during a 16-day growth experiment. These results demonstrate that rMTC 6-23 cells contain NT and suggest the possibility of an association between neurotensin and calcitonin.

Hyaluronidase-induced inhibition of the insulinotropic action of sulfonylureas, leucine, and glucagon in rodent isolated pancreatic islets.

Exposure of hamster pancreatic islets to hyaluronidase during isolation by means of collagenase inhibits the insulinotropic action of several chemically different sulfonylureas, leucine, and glucagon without affecting glucose-stimulated insulin secretion. This inhibition is reversible for tolbutamide and leucine but irreversible for glucagon. Hyaluronidase inhibits reversibly the insulinotropic action of tolbutamide without affecting that of glucose also in mouse and rat isolated pancreatic islets . These findings suggest the existence of functionally related pancreatic beta cell receptors for tolbutamide and leucine different from those for glucose and glucagon and illustrate the potential usefulness of hyaluronidase as an enzymatic probe applicable toward investigating the cellular mechanism of action of key insulinotropic agents.