Actions of rat growth hormone-releasing factor and norepinephrine on cytosolic free calcium and inositol trisphosphate in rat C-cells.

Rat growth hormone-releasing factor (rGRF) and norepinephrine (NE) stimulate secretion of calcitonin (CT) and neurotensin (NT) from cultured C-cells. The mechanism by which these agents cause secretion has not been well studied. We have examined the actions of the CT and NT secretagogues rGRF and NE on cytosolic free calcium concentrations ([Ca2+]i) in the rat C-cell line rMTC 44-2. Because inositol trisphosphate (IP3) has been shown to cause release of intracellular calcium stores in several cell types, we have also examined the effects of rat GRF, NE, and increases in extracellular calcium on IP3 accumulation in rMTC 44-2 cells. Stimulation by 10(-6) M rGRF caused a biphasic response in [Ca2+]i consisting of a rapid spike to 136 +/- 4% (mean +/- SE) of basal [Ca2+]i. This increase in [Ca2+]i decayed to base line and then gradually increased to 173 +/- 13% of basal [Ca2+]i. Stimulation by 10(-6) M NE gave a similar biphasic increase in [Ca2+]i. The increases in [Ca2+]i induced by both rGRF and NE were inhibited by pretreatment with EGTA or verapamil. rGRF, NE, and increasing concentrations of extracellular calcium, which all caused rapid increases in [Ca2+]i, failed to increase IP3 accumulation in rMTC 44-2 cells. These results suggest that rGRF- and NE-induced secretion in C-cells are mediated by changes in [Ca2+]i. These increases in [Ca2+]i appear to be generated by extracellular calcium influx rather than by release of intracellular calcium stores.

Measurement of cytosolic free Ca2+ concentrations in human and rat osteosarcoma cells: actions of bone resorption-stimulating hormones.

Influx of extracellular Ca++ into bone cells has been postulated as an early action of PTH and other bone resorption-stimulating factors. To test this hypothesis directly, we measured the cytosolic free Ca2+ concentration ([Ca2+]i) in two hormone-responsive human (SaOS-2 and G-292) and two rat osteosarcoma cell lines (Ros 25/1 and Ros 17/2.8) and in primary cultures of bone cells from neonatal mouse calvaria using the fluorescent Ca2+ indicator Quin 2. Actions of bovine PTH-(1-34), vasoactive intestinal peptide, epidermal growth factor, prostaglandin E2, and ionomycin were studied. Medium cAMP (20 min; 37 C; 25 microM 3-isobutyl-1-methylxanthine) was quantitated by RIA. Basal [Ca2+]i was: SaOS-2, 126 +/- 8 nM; G-292, 61 +/- 6 nM; Ros 25/1, 109 +/- 15 nM; Ros 17/2.8, 363 +/- 42 nM; and primary cultures, 266 +/- 39 nM (mean +/- SE; n = 3-14). In each cell type, no acute (1 sec to 20 min) spike in [Ca2+]i was observed in response to PTH (24-120 nM), vasoactive intestinal peptide (100 nM), epidermal growth factor (17 nM), or prostaglandin E2 (2.8 microM). However, in SaOS-2 cells only, PTH reproducibly increased [Ca2+]i 10-15% above basal values beginning about 3 min after hormone addition, and this small increase returned to baseline at 15-20 min. Ionomycin (100 nM) elicited an immediate spike in [Ca2+]i to levels 2- to 4-fold above basal in all cells; the peak [Ca2+]i decayed rapidly (within 4-5 min) to baseline in G-292, Ros 25/1, and Ros 17/2.8 cells. The decay of peak [Ca2+]i in SaOS-2 was prolonged. To test for intact hormone responses in Quin 2-loaded cells, cAMP accumulation was measured. In SaOS-2 and Ros 17/2.8, both control and Quin 2-loaded cells showed similar increases in cAMP in response to PTH. Considering the limitations of the Quin 2 technique, we conclude that in the four hormone-responsive bone cell lines and primary cultures of bone cells tested, acute elevation of [Ca2+]i is not an inevitable consequence of receptor occupancy and/or adenylate cyclase activation by bone resorption-stimulating hormones.

Evidence for multiple bone resorption-stimulating factors produced by normal human keratinocytes in culture.

Conditioned medium from cultured normal human foreskin keratinocytes enhanced the release of calcium from neonatal mouse calvaria in organ culture. Unfractionated keratinocyte-conditioned medium (KCM) stimulated bone resorption in a dose-dependent manner, but it did not increase the concentration of prostaglandin E2 (PGE2) in the bone culture medium until a maximal dose of KCM for resorption was used. Furthermore, inhibitors of PGE2 synthesis, indomethacin, ibuprofen, and piroxicam, did not inhibit KCM-induced calcium release. High concentrations of KCM increased cAMP production by calvaria in the presence of isobutylmethylxanthine, but the increase was small compared with that produced by a dose of bovine PTH that caused a similar level of bone resorption. The bone resorption-stimulating activity of KCM was not lost after incubation at 56 C for 60 min, but it was lost after heating at 100 C for 10 min. Fractionation of KCM by gel filtration chromatography revealed two distinct peaks of bone resorption-stimulating activity. One peak, KCMI, caused a significant increase in bone resorption at 2 micrograms protein/ml. KCMI did not increase medium PGE2, and inhibition of PGE2 synthesis in bone had no effect on KCMI-induced bone resorption. KCMI failed to increase cAMP production by human osteosarcoma SaOS-2 cells. Another peak, KCMII, caused a dose-dependent increase in bone resorption, and a significant increase in medium calcium was noted at a 20-fold lower concentration (0.1 microgram protein/ml) than with KCMI. In contrast to KCMI, the increase in bone resorption stimulated by KCMII was accompanied by a parallel increase in the production of PGE2, and inhibition of PGE2 synthesis completely inhibited the bone resorption-stimulating activity of KCMII. KCMII also caused an increase in cAMP production by SaOS-2 cells. We conclude that KCM contains at least two distinct bone resorption-stimulating factors, one of which acts via a PG-mediated mechanism and the other by a PG-independent pathway.

Two squamous cell carcinomas not associated with humoral hypercalcemia produce a potent bone resorption-stimulating factor which is interleukin-1 alpha.

Conditioned medium (CM) from two squamous cell carcinoma cell lines, SCC-9 and SCC-13, stimulated bone resorption in neonatal mouse calvariae in organ culture. Enhanced bone resorption induced by CM was associated with an increased production of prostaglandin-E2 (PGE2) by the calvariae. Complete inhibition of stimulated PGE2 synthesis by indomethacin only partially inhibited bone resorption-stimulating activity (BRSA) in the CM. Neither SCC-9 nor SCC-13 CM stimulated cAMP production in rat osteosarcoma cells (ROS 17/2.8). The BRSA in CM was completely inhibited by an antibody to interleukin-1 alpha (IL-1 alpha). Fractionation of SCC-9 CM by gel filtration and HPLC ion exchange chromatography revealed a single peak of BRSA and PGE2 synthesis-stimulating activity at 17-20K (termed SCMII). In mouse calvariae, SCMII increased medium Ca2+ and PGE2 in a dose-dependent manner at concentrations from 20 ng protein/ml to a maximum of 500 ng protein/ml. Preincubation of SCMII with antibody to IL-1 alpha completely inhibited SCMII-induced bone resorption. SCMII also enhanced thymocyte proliferation with activity that was equivalent to 353 U/ml IL-1. Antibodies to IL-1 beta and tumor necrosis factor had no effect on SCMII-induced bone resorption. Using specific enzyme-linked immunosorbent assays for IL-1 alpha and IL-1 beta, IL-1 alpha was measured in high concentrations in both crude and partially purified fractions of SCC-9 and SCC-13 CM. In contrast, IL-1 beta was either undetectable or present in amounts below those that stimulate bone resorption. In addition, SCMII did not enhance cAMP production in bone cells. We conclude that the BRSA produced by the two squamous cell carcinoma cell lines SCC-9 and SCC-13 is IL-1 alpha.

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells.

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.

Serological detection of common human melanoma membrane antigens by microcomplement fixation and immunofluorescence.

An antiserum to human melanoma antigens was obtained from a melanoma patient after immunization with autologous irradiated cultured melanoma cells and bacillus Calmette-Guerin. Using the microcomplement fixation assay, the antiserum, at a titer of 1/1,800, was noted to bind strongly with 7 of 10 allogeneic cultured human melanoma cells lines. However, using the indirect immunofluorescence test and serum at a much lower titer (1/8), only 3 of the 10 melanoma cell lines were positive. Using both microcomplement fixation and indirect immunofluorescence, no significant reactivity was noted in several nonmelanoma cell lines including Hela, human lung adenocarcinoma, human prostatic carcinoma, WI-38 and VA-13 cell lines. These data suggest that common melanoma membrane antigens exist on 7 of 10 cultured human melanoma cell lines as tested by microcomplement fixation and that this assay is more sensitive than immunofluorescence. These common melanoma membrane antigens may eventually be extracted, purified and used for specific immunodiagnosis and immunotherapy.